2. The conjugate of claim 1 wherein the growth hormone conjugate has the
formula (I): A-W-B-GH (I) wherein: GH is a growth hormone compound, B
is a hydrophilic spacer, W is a chemical group linking A and B, and A is
an albumin binding residue;

7. The conjugate of claim 4, wherein D1 and D2 are independently selected
from --O-- or --NR6-- or a valence bond.

8. The conjugate of claim 4, wherein E1 and E2 are independently selected
from --O-- or --NR6-- or a valence bond.

9. The conjugate of claim 4, wherein W1 through W8
independently are selected from the group consisting of --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- and a valence bond.

10. The conjugate of claim 4, wherein R1, R2, R3, R4
and R5 independently are selected from the group consisting of
hydrogen, --C(O)OH, --C(O)NH2, --S(O)2OH and C1-6-alkyl,
wherein the alkyl group optionally is substituted with --C(O)OH,
--C(O)NH2, or --S(O)2OH.

11. The conjugate of claim 2, wherein A is selected from ##STR00086##
##STR00087## wherein * denotes the attachment to B through W.

12. The conjugate of claim 1, wherein the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the position
corresponding to position 40 in SEQ ID No. 1, or the glutamine residue in
the position corresponding to position 141 in SEQ ID No. 1, or the
N-terminal residue of the growth hormone compound.

13. The conjugate of claim 1, wherein said compound is selected from the
group consisting of ##STR00088## ##STR00089## ##STR00090##
##STR00091## ##STR00092## ##STR00093##

14. A pharmaceutical composition comprising a conjugate of claim
1,optionally in combination with a pharmaceutically acceptable excipient.

15. A method of treating one a disease or condition treatable with an
increased amount of circulating growth hormone comprising administering
an effective amount of the growth hormone conjugate of claim 1.

16. The method of claim 15 wherein the disease or condition is selected
from the group consisting of growth hormone deficiency (GHD), Turner
Syndrome, Prader-Willi syndrome (PWS), Noonan syndrome and idiopathic
short stature (ISS).

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to a growth hormone compound linked
to an albumin binding residue via a hydrophilic spacer, and to methods of
preparing and using such compounds. These compounds have a protracted
profile of action and are useful in therapy.

BACKGROUND OF THE INVENTION

[0002] It is well-known to modify the properties and characteristics of
peptides by conjugating groups to the peptide which duly changes the
properties of the peptide. Such conjugation generally requires some
functional group in the peptide to react with another functional group in
a conjugating group. Typically, amino groups, such as the N-terminal
amino group or the ε-amino group in lysines, have been used in
combination with a suitable acylating reagent such as described for GLP-1
in WO2005/027978. Alternatively, polyethylene glycol (PEG) or derivatives
thereof may be attached to proteins. For a review, see Exp. Opion. Ther.
Patent., 14, 859-894, (2004). It has been shown that the attachment of
PEG to growth hormone may have a positive effect on the plasma half-life
of growth hormone, WO 03/044056.

[0003] The use of carboxypeptidases to modify the C-terminal of peptides
has been described earlier. WO 92/05271 discloses the use of
carboxypeptidases and nucleophilic compounds to amidate the C-terminal
carboxy group, and WO 98/38285 discloses variants of carboxypeptidase Y
particular suitable for this purpose.

[0004] EP 243 929 discloses the use of carboxypeptidase to incorporate
polypeptides, reporter groups or cytotoxic agents into the C-terminal of
proteins or polypeptides.

[0005] WO 2005/035553 describes methods for selective conjugation of
peptides by enzymatically incorporating a functional group at the
C-terminal of a peptide.

[0006] Transglutaminase has previously been used to alter the properties
of peptides. In the food industry and particular in the diary industry
many techniques are available to e.g. cross-bind peptides using
transglutaminases. Other documents disclose the use of transglutaminase
to alter the properties of physiologically active peptides. EP 950665, EP
785276 and Sato, Adv. Drug Delivery Rev. 54, 487-504 (2002) disclose the
direct reaction between peptides comprising at least one Gln and
amine-functionalised PEG or similar ligands in the presence of
transglutaminase, and Wada, Biotech. Lett. 23, 1367-1372 (2001) discloses
the direct conjugation of β-lactoglobulin with fatty acids by means
of transglutaminase. The international patent application published as WO
2005/070468 discloses the use of transglutaminase to incorporate a handle
whereto conjugating groups can be attached.

[0007] Growth hormone is a key hormone involved in the regulation of not
only somatic growth, but also in the regulation of metabolism of
proteins, carbohydrates and lipids. The major effect of growth hormone is
to promote growth. Human growth hormone is a 191 amino acid residue
protein with the sequence:

[0008] Administration of human growth hormone and closely related variants
thereof is used to treat a variety of growth hormone deficiency related
diseases. Being a peptide, growth hormone is administered parenterally,
i.e., by means of a needle. Growth hormone is, furthermore, characterised
by a relative short half-life, hence frequent administrations are
required with the corresponding pain and inconvenience for the patient.
Hence, there is still a need for the provision of growth hormone
compounds with improved pharmacological properties, such as e.g.
prolonged half-life.

[0009] The present invention provides novel growth hormone conjugates with
improved pharmacological properties as well as methods for their
production.

SUMMARY OF THE INVENTION

[0010] The bioavailability of a subcutaneously administered pharmaceutical
compound may be related to the absorption rate. The ability of a compound
to pass the tight junctions of the subcutaneous capillaries may in part
be related to their physical and chemical properties as well as the
molecular size or the hydrodynamic volume of the compound. A protein
conjugate such as a pegylated hGH (PEG-hGH) with a 40 kDa PEG has an
apparent molecular weight of 150-250 kDa. A hGH molecule with covalent
bound albumin has a molecular weight of 87 kDa, whereas a hGH molecule
with a non-covalent bound albumin will be dissociated from albumin part
of the time and thus have a molecular weight of 22 kDa.

[0011] It is contemplated that the time spend in the dissociated state
depends, at least partly, on the affinity of the albumin binding moiety.
Thus the absorption rate of a hGH molecule with a non-covalent bound
albumin may be faster than for a PEG-hGH. An increased rate of absorption
may be obtained when using albumin binding moieties having lower affinity
for albumin.

[0012] Additionally, the physical and chemical properties of the linker
and/or the spacer providing the attachment of the albumin binding moiety
to hGH will influence the functionalities of the compounds.

[0013] The present inventors have surprisingly found that growth hormone
compounds (GH) may be selectively linked to an albumin binding
residue--via a hydrophilic spacer that separates the GH and the albumin
binding residue with a chemical moiety having a mLogP<0--or a
cLogP<0.5 to obtain GH conjugates with improved pharmacological
properties.

[0014] Furthermore, the present invention is based on the observation that
introducing an albumin binding residue via a hydrophilic spacer in human
growth hormone (hGH) can be done selectively wherein a large proportion
of the biological activity can been retained. Preferably, an albumin
binding residue via a hydrophilic spacer is introduced at the positions
corresponding to positions phenylalanine 1, glutamine 40 and/or glutamine
141 in hGH having the sequence of SEQ ID No.1. The use of
transglutaminase (TGase), and in particular TGase from
Streptoverticillium mobaraenae or Streptomyces lydicus allows a selective
introduction of an albumin binding residue via a hydrophilic spacer at
positions 40 and/or 141, and the remaining 11 glutamine residues are left
untouched despite the fact that glutamine is a substrate for
transglutaminase.

[0015] Thus, in one embodiment of the present invention the growth hormone
compound (GH) is linked to one albumin binding residue via a hydrophilic
spacer. Typically, the albumin binding residue is attached to the
N-terminal, position 40 or position 141 of hGH via a hydrophilic spacer.
In further embodiments two or three albumin binding residues are attached
to the N-terminal, position 40 and/or position 141 of hGH via a
hydrophilic spacer.

[0016] In a further embodiment of the present invention the hydrophilic
spacer has the formula

[0029] It is a still further objective of the present invention to provide
a method for improving the properties of a GH by conjugation said protein
according to the methods of the present invention.

DEFINITIONS

[0030] In the present context, the term "growth hormone compound" as used
herein means growth hormone of mammalian origin, such as human, bovine,
or porcine growth hormone, and recombinant growth hormone, such as
recombinant human, bovine, or porcine growth hormone, and variants of
such growth hormones. As used herein "GH" and "growth hormone compound"
are interchangeable. When GH is a variant of growth hormone of mammalian
origin, such as hGH and recombinant hGH, said variant is understood to be
the compound obtained by substituting one or more amino acid residues in
the growth hormone, e.g. hGH, sequence with another natural or unnatural
amino acid; and/or by adding one or more natural or unnatural amino acids
to the growth hormone, e.g. hGH, sequence; and/or by deleting one or more
amino acid residue from the growth hormone, e.g. hGH, sequence, wherein
any of these steps may optionally be followed by further derivatization
of one or more amino acid residues. In particular, such substitutions are
conservative in the sense that one amino acid residue is substituted by
another amino acid residue from the same group, i.e. by another amino
acid residue with similar properties. Amino acids may conveniently be
divided in the following groups based on their properties: Basic amino
acids (such as arginine, lysine, histidine), acidic amino acids (such as
glutamic acid and aspartic acid), polar amino acids (such as glutamine,
cysteine and asparagine), hydrophobic amino acids (such as leucine,
isoleucine, proline, methionine and valine), aromatic amino acids (such
as phenylalanine, tryptophan, tyrosine) and small amino acids (such as
glycine, alanine, serine and threonine). Typically, the GH has at least
80% identity with hGH, and typically, has at least 20% of the growth
hormone activity of hGH as determined in assay I herein.

[0031] In the present context, the term "albumin binding residue" as used
herein means a residue which binds noncovalently to human serum albumin.
The albumin binding residue attached to the growth hormone compound (GH)
typically has a binding affinity towards human serum albumin that is
below about 10 μM or even below about 1 μM. A range of albumin
binding residues are known among linear and branched lipohophillic
moieties containing 12-40 carbon atoms, compounds with a
cyclopentanophenanthrene skeleton, and/or peptides having 10-45 amino
acid residues etc. Albumin binding properties can be measured by surface
plasmon resonance as described in J. Biol. Chem. 277(38), 35035-35042,
(2002).

[0032] The term "hydrophilic spacer" as used herein means a spacer that
separates a growth hormone compound and an albumin binding residue with a
chemical moiety which comprises at least 5 nonhydrogen atoms where 30-50%
of these are either N or O.

[0033] In the present context, the term "transamination" and related terms
are intended to indicate a reaction wherein the amide nitrogen in the
side chain of glutamine is exchanged with nitrogen from another compound,
in particular nitrogen from another nitrogen containing nucleophile.

[0034] Transglutaminase (E.C.2.3.2.13) is also known as
protein-glutamine-γ-glutamyltransferase and catalyses the general
reaction

##STR00001##

Q--C(O)--NH2 (amine acceptor) may represent a glutamine residue
containing peptide or protein and Q'--NH2 (amine donor) represents
an amine-containing nucleophile. Alternatively, Q--C(O)--NH2 and
Q'--NH2 may represent an amine acceptor and a lysine-containing
peptide or protein, respectively. In the present invention, however,
Q--C(O)--NH2 represents a glutamine residue containing growth
hormone and Q'--NH2 represents an amine-containing nucleophile as
indicated above.

[0035] Examples of useful transglutaminases include microbial
transglutaminases, such as e.g. those from Streptomyces mobaraense,
Streptomyces cinnamoneum and Streptomyces griseocarneum (all disclosed in
U.S. Pat. No. 5,156,956, which is incorporated herein by reference), and
from Streptomyces lavendulae (disclosed in U.S. Pat. No. 5,252,469, which
is incorporated herein by reference) and Streptomyces ladakanum (JP
2003/199569, which is incorporated herein by reference). It should be
noted that members of the former genus Streptoverticillium are now
included in the genus Streptomyces (Kaempfer, J. Gen. Microbiol. 137,
1831-1892 (1991)). Other useful microbial transglutaminases have been
isolated from Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183,
which is incorporated herein by reference) and from various Myxomycetes.
Other examples of useful microbial transglutaminases are those disclosed
in WO 96/06931 (e.g. transglutaminase from Bacillus lydicus) and WO
96/22366, both of which are incorporated herein by reference. Useful
non-microbial transglutaminases include guinea-pig liver
transglutaminase, and transglutaminases from various marine sources like
the flat fish Pagrus major (disclosed in EP-0555649, which is
incorporated herein by reference), and the Japanese oyster Crassostrea
gigas (disclosed in U.S. Pat. No. 5,736,356, which is incorporated herein
by reference).

[0036] In the present context, the term "not accessible" is intended to
indicate that something is absent or de facto absent in the sense that it
cannot be reached. When it is stated that functional groups are not
accessible in a protein to be conjugated it is intended to indicate that
said functional group is absent from the protein or, if present, in some
way prevented from taking part in reactions. By way of example, said
functional group could be buried deep in the structure of the protein so
that it is shielded from participating in the reaction. It is recognised
that whether or not a functional group is accessible depends on the
reaction conditions. It may be envisaged that, e.g. in the presence of
denaturing agents or at elevated temperatures the protein may unfold to
expose otherwise not accessible functional groups. It is to be understood
that "not accessible" means "not accessible at the reaction condition
chosen for the particular reaction of interest".

[0037] The term "alkane" or "alkyl" is intended to indicate a saturated,
linear, branched and/or cyclic hydrocarbon. Unless specified with another
number of carbon atoms, the term is intended to indicate hydrocarbons
with from 1 to 30 (both included) carbon atoms, such as 1 to 20 (both
included), such as from 1 to 10 (both included), e.g. from 1 to 5 (both
included). The terms alkyl and alkylene refer to the corresponding
radical and bi-radical, respectively.

[0038] The term "C1-6 alkyl" refers to a straight chained or branched
saturated hydrocarbon having from one to six carbon atoms inclusive.
Examples of such groups include, but are not limited to, methyl,
2-propyl, 1-butyl, 2-butyl, 2-methyl-2-propyl, 2-methyl-1-butyl and
n-hexyl.

[0040] The term "alkene" is intended to indicate linear, branched and/or
cyclic hydrocarbons comprising at least one carbon-carbon double bond.
Unless specified with another number of carbon atoms, the term is
intended to indicate hydrocarbons with from 2 to 30 (both included)
carbon atoms, such as 2 to 20 (both included), such as from 2 to 10 (both
included), e.g. from 2 to 5 (both included). The terms alkenyl and
alkenylene refer to the corresponding radical and bi-radical,
respectively.

[0041] The term "alkyne" is intended to indicate linear, branched and/or
cyclic hydrocarbons comprising at least one carbon-carbon triple bond,
and it may optionally comprise one or more carbon-carbon double bonds.
Unless specified with another number of carbon atoms, the term is
intended to indicate hydrocarbons with from 2 to 30 (both included)
carbon atoms, such as from 2 to 20 (both included), such as from 2 to 10
(both included), e.g. from 2 to 5 (both included). The terms alkynyl and
alkynylene refer to the corresponding radical and bi-radical,
respectively.

[0042] The term "homocyclic aromatic compound" is intended to indicate
aromatic hydrocarbons, such as benzene and naphthalene.

[0044] The terms "hetero alkane", "hetero alkene" and "hetero alkyne" are
intended to indicate alkanes, alkenes and alkynes as defined above, in
which one or more hetero atom or group have been inserted into the
structure of said moieties. Examples of hetero groups and atoms include
--O--, --S--, --S(O)--, --S(O)2--, --C(O)--C(S)-- and --N(R*)--,
wherein R* represents hydrogen or C1-C6-alkyl. Examples of
heteroalkanes include.

##STR00002##

[0045] The term "radical" or "biradical" is intended to indicate a
compound from which one or two, respectively, hydrogen atoms have been
removed. When specifically stated, a radical may also indicate the moiety
formed by the formal removal of a larger group of atoms, e.g. hydroxyl,
from a compound.

[0046] The term "halogen" is intended to indicate members of the seventh
main group of the periodic table, e.g. F, Cl, Br and I.

[0047] In the present context, the term "aryl" is intended to indicate a
carbocyclic aromatic ring radical or a fused aromatic ring system radical
wherein at least one of the rings are aromatic. Typical aryl groups
include phenyl, biphenylyl, naphthyl, and the like.

[0048] The term "heteroaryl" or "hetaryl", as used herein, alone or in
combination, refers to an aromatic ring radical with for instance 5 to 7
member atoms, or to a fused aromatic ring system radical with for
instance from 7 to 18 member atoms, wherein at least one ring is
aromatic, containing one or more heteroatoms as ring atoms selected from
nitrogen, oxygen, or sulfur heteroatoms, wherein N-oxides and sulfur
monoxides and sulfur dioxides are permissible heteroaromatic
substitutions. Examples include furanyl, thienyl, thiophenyl, pyrrolyl,
imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl,
isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridinyl,
pyridazinyl, pyrazinyl, pyrimidinyl, quinolinyl, isoquinolinyl,
benzofuranyl, benzothiophenyl, indolyl, and indazolyl, and the like.

[0049] The term "conjugate" as a noun is intended to indicate a modified
protein, i.e. a protein with a moiety bonded to it in order to modify the
properties of said protein. As a verb, the term is intended to indicate
the process of bonding a moiety to a protein to modify the properties of
said protein.

[0050] As used herein, the term "prodrug" indicates biohydrolyzable amides
and biohydrolyzable esters and also encompasses a) compounds in which the
biohydrolyzable functionality in such a prodrug is encompassed in the
compound according to the present invention, and b) compounds which may
be oxidized or reduced biologically at a given functional group to yield
drug substances according to the present invention. Examples of these
functional groups include 1,4-dihydropyridine,
N-alkylcarbonyl-1,4-dihydropyridine, 1,4-cyclohexadiene, tert-butyl, and
the like.

[0051] As used herein, the term "biohydrolyzable ester" is an ester of a
drug substance (in casu, a compound according to the invention) which
either a) does not interfere with the biological activity of the parent
substance but confers on that substance advantageous properties in vivo
such as duration of action, onset of action, and the like, or b) is
biologically inactive but is readily converted in vivo by the subject to
the biologically active principle. The advantage is, for example
increased solubility or that the biohydrolyzable ester is orally absorbed
from the gut and is transformed to a compound according to the present
invention in plasma. Many examples of such are known in the art and
include by way of example lower alkyl esters (e.g., C1-C4),
lower acyloxyalkyl esters, lower alkoxyacyloxyalkyl esters, alkoxyacyloxy
esters, alkyl acylamino alkyl esters, and choline esters.

[0052] As used herein, the term "biohydrolyzable amide" is an amide of a
drug substance (in casu, a compound according to the present invention)
which either a) does not interfere with the biological activity of the
parent substance but confers on that substance advantageous properties in
vivo such as duration of action, onset of action, and the like, or b) is
biologically inactive but is readily converted in vivo by the subject to
the biologically active principle. The advantage is, for example
increased solubility or that the biohydrolyzable amide is orally absorbed
from the gut and is transformed to a compound according to the present
invention in plasma. Many examples of such are known in the art and
include by way of example lower alkyl amides, a-amino acid amides,
alkoxyacyl amides, and alkylaminoalkylcarbony) amides.

[0054] A "therapeutically effective amount" of a compound as used herein
means an amount sufficient to cure, alleviate or partially arrest the
clinical manifestations of a given disease and its complications. An
amount adequate to accomplish this is defined as "therapeutically
effective amount". Effective amounts for each purpose will depend on the
severity of the disease or injury as well as the weight and general state
of the subject. It will be understood that determining an appropriate
dosage may be achieved using routine experimentation, by constructing a
matrix of values and testing different points in the matrix, which is all
within the ordinary skills of a trained physician or veterinary.

[0055] The term "treatment" and "treating" as used herein means the
management and care of a patient for the purpose of combating a
condition, such as a disease or a disorder. The term is intended to
include the full spectrum of treatments for a given condition from which
the patient is suffering, such as administration of the active compound
to alleviate the symptoms or complications, to delay the progression of
the disease, disorder or condition, to alleviate or relief the symptoms
and complications, and/or to cure or eliminate the disease, disorder or
condition as well as to prevent the condition, wherein prevention is to
be understood as the management and care of a patient for the purpose of
combating the disease, condition, or disorder and includes the
administration of the active compounds to prevent the onset of the
symptoms or complications. The patient to be treated is preferably a
mammal; in particular a human being, but it may also include animals,
such as dogs, cats, cows, sheep and pigs.

DESCRIPTION OF THE INVENTION

[0056] In its broadest aspect the present invention relates to a growth
hormone conjugate which comprises a growth hormone compound (GH) linked
selectively to an albumin binding residue via a hydrophilic spacer, or a
pharmaceutically acceptable salt, solvate or prodrug thereof.

[0057] In one embodiment of the present invention the hydrophilic spacer
has a mLogP<0.

[0058] Solubility of a hydrophilic spacer can be described by its logP
value. LogP, also known as the partition coefficient, is the logarithm of
the ratio of concentrations of a compound in the two phases of a mixture
of two immiscible solvents at equilibrium. Typically one of the solvents
is water while the second is selected from octan-1-ol, chloroform,
cyclohexane and propylene glycol dipelargonate (PGDP). LogP values
measured in these different solvents show differences principally due to
hydrogen bonding effects. Octanol can donate and accept hydrogen bonds
whereas cyclohexane is inert. Chloroform can donate hydrogen bonds
whereas PGDP can only accept them.

[0059] In another embodiment of the invention, the hydrophilic spacer has
a LogP of below -0.5 in either octan-1-ol, chloroform, cyclohexane and
propylene glycol dipelargonate (PGDP).

[0060] In a further embodiment, the hydrophilic spacer has a logP below -1
in either octan1-ol, chloroform, cyclohexane and propylene glycol
dipelargonate (PGDP).

[0061] Alternatively, the LogP value can be calculated as mLogP and/or
cLogP for the albumin binder part or hydrophilic spacer part using
published algorithms (J. Am. Chem. Soc., 86 (1964) 5175-5180 "A New
Substituent Constant, , Derived from Partition Coefficients", C. A.
Lipinski et al. Advanced Drug Delivery Reviews, 23 (1997) 3-25,
"Experimental and Computational Approaches to Estimate Solubility and
Permeability in Drug Discovery and Development Settings" and I.
Moriguchi, S. Hirono, I. Nakagome, H. Hirano, Chem. and Pharm. Bull., 42
(1994) 976-978 "Comparison of Reliability of logP Values for Drugs
Calculated by Several Methods". In one embodiment of the present
invention the hydrophilic spacer has a cLogP<0.5.

[0062] In a further embodiment the growth hormone compound (GH) is linked
to one albumin binding residue via a hydrophilic spacer.

[0063] In another embodiment the growth hormone compound (GH) is linked to
two albumin binding residues via one or two hydrophilic spacer(s). Thus,
in one example one albumin binding residue is linked via one hydrophilic
spacer to glutamine in position 40 and the other albumin binding residue
is linked via one hydrophilic spacer to glutamine in position 141; or
alternatively two albumin binding residues are linked via one hydrophilic
spacer to glutamine in position 40 or 141 or the N-terminal. In a still
other embodiment the growth hormone compound (GH) is linked to three
albumin binding residues via one or more hydrophilic spacer(s).

[0064] In an embodiment the hydrophilic spacer comprise at least one OEG
motif, the radical 8-amino-3,6-dioxaoctanic acid, i.e.
--NH--(CH2)2--O--(CH2)2--O--CH2--C(O)--. In
further specified embodiment the hydrophilic spacer comprise at least two
OEG motifs. The orientation of such OEG motif(s) is in one embodiment so
that the --C(O)-- is closest to the growth hormone compound but not
connecting the growth hormone compound and the albumin binder linker and
the --NH-- is closest to the albumin binding residue. In additional
embodiments comprising two OEG motifs the two motifs have identical
orientation or different orientation. In an embodiment two such OEG
motifs are located adjacent to each other whereas in alternative
embodiments such OEG motifs are serrated by one or more atoms covalently
linked.

[0065] In an embodiment the hydrophilic spacer comprise at lease one
glutamic acid residue. The amino acid glutamic acid comprises two
carboxylic acid groups. Its gamma-carboxy group may be used for forming
an amide bond with the epsilon-amino group of lysine, or with an amino
group of an OEG molecule, if present, or with the amino group of another
Glu residue, if present. The alfa-carboxy group may alternatively be used
for forming similar amide bond with the epsilon-amino group of lysine, or
with an amino group of an OEG molecule, if present, or with the amino
group of another Glu residue, if present. The amino group of Glu may in
turn form an amide bond with the carboxy group of the albumin binding
residue, or with the carboxy group of an OEG motif, if present, or with
the gamma-carboxy group or alfa carboxy group of another Glu, if present.
The linkage of the amino group of one Glu to a gamma carboxy group of a
second Glu may be referred to as a "gamma-Glu" motif.

[0066] In an embodiment the hydrophilic spacer comprise at lease one
combined OEG-Glu motif
(--NH--(CH2)2--O--(CH2)2--O--CH2--C(O)NH-CH(C(O)-
OH)--(CH2)2--C(O)--) or at least one combined Glu-OEG motif
(--NH--CH(C(O)OH)--(CH2)2--C(O)NH--(CH2)2--O--(CH.sub-
.2)2--O--CH2--C(O)--) or combination here of, where in such
Glu-OEG and OEG-Glu motifs may be separated by one or more covalently
linked atoms or directly bond to each other by an amide bond of the Glu's
forming a gamma-Glu.

[0067] In a still further embodiment of the growth hormone conjugate the
hydrophilic spacer has the formula

[0080] In an embodiment D1 is selected from --O--, --NR6--,
--N(COR7)-- or a valence bond, D2 is --NR6--, E1 and E2 are
independently selected from --O--, --NR6--, --N(COR7)-- or a
valence bond and R6 and R7 independently represent hydrogen or
C1-6-alkyl,

[0081] In a further aspect the present invention relates to a growth
hormone conjugate of the formula (I):

[0087] As described above the growth hormone compound (GH) may be linked
to two albumin binding residues via a hydrophilic spacer, accordingly in
a still further aspect the present invention relates to a growth hormone
conjugate of the formula (II):

[0094] In the conjugate of formula (II) W' is selected from the same
groups as W, A' is selected from the same groups as A and B' is selected
from the same groups as B, and it should be understood that W and W', A
and A', and B and B' are independently selected from any one of the
respective groups as defined hereunder. Thus, any embodiments of W, A,
and B hereunder are also embodiments of W', A', and B'. Furthermore, any
one of the embodiments described herein refers independently to both of
the conjugates of formula (I) and (II), as well as the broad aspect and
embodiments thereof when suitable.

[0095] The above embodiments as well as the embodiments to be described
hereunder should be seen as referring to any one of the aspects described
herein as well as any one of the embodiments described herein unless it
is specified that an embodiment relates to a certain aspect or aspects of
the present invention.

[0096] In one embodiment GH is a variant of hGH, wherein a variant is
understood to be the compound obtained by substituting one or more amino
acid residues in the hGH sequence with another natural or unnatural amino
acid; and/or by adding one or more natural or unnatural amino acids to
the hGH sequence; and/or by deleting one or more amino acid residue from
the hGH sequence, wherein any of these steps may optionally be followed
by further derivatization of one or more amino acid residues. In
particular, such substitutions are conservative in the sense that one
amino acid residue is substituted by another amino acid residue from the
same group, i.e. by another amino acid residue with similar properties.
Amino acids may conveniently be divided in the following groups based on
their properties: Basic amino acids (such as arginine, lysine,
histidine), acidic amino acids (such as glutamic acid and aspartic acid),
polar amino acids (such as glutamine, cysteine and asparagine),
hydrophobic amino acids (such as leucine, isoleucine, proline, methionine
and valine), aromatic amino acids (such as phenylalanine, tryptophan,
tyrosine) and small amino acids (such as glycine, alanine, serine and
threonine.).

[0097] In a further embodiment GH represents a growth hormone compound
comprising an amino acid sequence having at least 90% identity to the
amino acid sequence of human growth hormone (hGH) (SEQ ID NO:1). In
further embodiments, GH has at least 80%, such as at least 85%, such as
at least 95% identity with hGH. In further embodiments, said identities
to hGH is coupled to at least 20%, such as at least 40%, such as at least
60%, such as at least 80% of the growth hormone activity of hGH as
determined in assay I herein. Any one of the sequence identity
embodiments may be combined with any one of the activity embodiments ,
such as a GH having at least 80% identity with hGH and coupled to at
least 60% of the growth hormone activity of hGH; a GH having at least 90%
identity with hGH and coupled to at least 40% of the growth hormone
activity of hGH; a GH having at least 95% identity with hGH and coupled
to at least 80% of the growth hormone activity of hGH, and so forth.

[0098] In a still further embodiment GH is hGH (SEQ ID NO:1).

[0099] In a further embodiment of the conjugate of formula (I) or (II),
the hydrophilic spacer B has the formula

[0112] In a further embodiment W1 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W1 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2--,

[0113] In a further embodiment W2 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W2 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2--,

[0114] In a further embodiment W3 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W3 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2--,

[0115] In a further embodiment W4 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W4 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2--,

[0116] In a further embodiment W5 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W5 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2--,

[0117] In a further embodiment W6 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W6 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2--,

[0118] In a further embodiment R1 selected from hydrogen, --C(O)OH,
--C(O)NH2, --S(O)2OH or C1-6-alkyl; wherein the alkyl
group optionally is substituted with --C(O)OH, --C(O)NH2,
--S(O)2OH. Typically, R1 is selected from --C(O)OH,
--C(O)NH2, or C1-6-alkyl; wherein the alkyl group optionally is
substituted with --C(O)OH, --C(O)NH2, or --S(O)2OH.

[0119] In a further embodiment R2 is selected from hydrogen,
--C(O)OH, --C(O)NH2, --S(O)2OH or C1-6-alkyl; wherein the
alkyl group optionally is substituted with --C(O)OH, --C(O)NH2,
--S(O)2OH. Typically, R2 is selected from --C(O)OH,
--C(O)NH2, or C1-6-alkyl; wherein the alkyl group optionally is
substituted with --C(O)OH, --C(O)NH2, or --S(O)2OH.

[0120] In a further embodiment R3 is selected from hydrogen,
--C(O)OH, --C(O)NH2, --S(O)2OH or C1-6-alkyl; wherein the
alkyl group optionally is substituted with --C(O)OH, --C(O)NH2,
--S(O)2OH. Typically, R3 is selected from --C(O)OH,
--C(O)NH2, or C1-6-alkyl; wherein the alkyl group optionally is
substituted with --C(O)OH, --C(O)NH2, or --S(O)2OH.

[0121] In a further embodiment R4 is selected from hydrogen,
--C(O)OH, --C(O)NH2, --S(O)2OH or C1-6-alkyl; wherein the
alkyl group optionally is substituted with --C(O)OH, --C(O)NH2,
--S(O)2OH. Typically, R4 is selected from --C(O)OH,
--C(O)NH2, or C1-6-alkyl; wherein the alkyl group optionally is
substituted with --C(O)OH, --C(O)NH2, or --S(O)2OH.

[0122] In a further embodiment R5 is selected from hydrogen,
--C(O)OH, --C(O)NH2, --S(O)2OH or C1-6-alkyl; wherein the
alkyl group optionally is substituted with --C(O)OH, --C(O)NH2,
--S(O)2OH. Typically, R5 is selected from --C(O)OH,
--C(O)NH2, or C1-6-alkyl; wherein the alkyl group optionally is
substituted with --C(O)OH, --C(O)NH2, or --S(O)2OH.

[0123] In a further embodiment E1 and E2 independently are selected from
--O--, --NR6--, --N--(COR7)-- or a valence bond. R6 and
R7 independently represent hydrogen or C1-6-alkyl.

[0124] In a further embodiment E1 is selected from --O-- or --NR6--
or a valence bond. Typically, E1 is selected from --O--.

[0125] In a further embodiment E2 is selected from --O-- or --NR6--
or a valence bond. Typically, E2 is selected from --O--.

[0126] In a further embodiment E1 and E2 are both --O--.

[0127] In a further embodiment E1 and E2 are both --NR6--.

[0128] In a further embodiment F is phenyl, pyrrolidine-2,5-dione or a
valence bond.

[0129] In a further embodiment D1 is selected from --O--, --NR6--,
--N(COR7)-- or a valence bond.

[0130] In a further embodiment D1 is selected from --O-- or --NR6--
or a valence bond. Typically, D1 is selected from --NR6--.

[0131] In a further embodiment D2 is selected from --O--, --NR6--,
--N(COR7)-- or a valence bond.

[0132] In a further embodiment D2 is selected from --O-- or --NR6--
or a valence bond. Typically, D2 is selected from --NR6--.

[0133] R6 and R7 independently represent hydrogen or
C1-6-alkyl,

[0134] In a further embodiment I1 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

[0135] In a further embodiment I2 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

[0136] In a further embodiment I3 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

[0137] In a further embodiment I4 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

[0138] In a further embodiment I5 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

[0139] In a further embodiment I6 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

[0140] In a further embodiment m1 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

[0141] In a further embodiment m2 is 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10.

[0142] In a further embodiment m3 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

[0143] In a further embodiment m4 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

[0144] In a further embodiment m5 is 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10.

[0145] In a further embodiment m6 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

[0146] In a further embodiment m7 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

[0147] In a further embodiment n1 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

[0148] In a further embodiment n2 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

[0149] In a further embodiment n3 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

[0150] In a further embodiment n4 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

[0151] In a further embodiment X1 is
--W1--[(CHR1).sub.I1--W2]m1--{[(CH2)n1O].su-
b.m2--[(CHR2).sub.I2--W3]m3}n2-- and X2 is
--[(CHR3).sub.I3--W4]m4--{[(CH2)n3O]m5--[(C-
HR4).sub.I4--W5}n4--, wherein
--{[(CH2)n1O]m2--[(CHR2)Is--W3]m3}.sub-
.n2-- and --{[(CH2)n3O]m5--[(CHR4).sub.I4--W5].su-
b.m6}n4-- are selected from,

##STR00003##

[0152] wherein * is intended to denote a point of attachment, ie, an open
bond.

[0153] In a further embodiment the molar weight of said hydrophilic spacer
is in the range from 80 Daltons (D) to 1500 D or in the range from 500 D
to 1100 D.

[0162] In an embodiment W7 is selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W7 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2.

[0163] In a further embodiment W8 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. Typically, W8 is selected
from --C(O)NH--, --NHC(O)--, --C(O)NHS(O)2.

[0164] In a further embodiment I7 is 0 or 1.

[0165] In a further embodiment the hydrophilic spacer B of the present
invention is selected from

##STR00004## ##STR00005## ##STR00006## ##STR00007##

wherein * is intended to denote a point of attachment, ie, an open bond.

[0166] The albumin binding residue (substituent A in formula (I) or (II)
above) attached to the growth hormone compound of the present invention
is a lipophilic residue, which binds non-covalently to albumin.
Typically, albumin binding residue is negatively charged at physiological
pH, and has a binding affinity towards human serum albumin that is below
about 10 μM or even below about 1 μM.

[0167] In a further embodiment of the growth hormone compound of the
present invention the albumin binding residue is selected from a straight
chain alkyl group, a branched alkyl group, a group which has a
w-carboxylic acid group or a w-carboxylic acid isoster. Typically, the
albumin binding residue has from 6 to 40 carbon atoms. In a further
embodiment the albumin binding residue has from 8 to 26 carbon atoms. In
a further embodiment the albumin binding residue has from 8 to 20 carbon
atoms.

[0168] In a further embodiment A has 14 to 26 carbon atoms and comprises
an ω-carboxylic acid group. In a further embodiment A has 14 to 26
carbon atoms and comprises an ω-carboxylic acid isoster, such as a
tetrazol.

[0169] In a further embodiment A is selected from

##STR00008## ##STR00009##

[0170] wherein * denotes the attachment to B through W.

[0171] The hydrophilic spacer (B) is preferably introduced in a position
of the growth hormone compound (GH) in a selective manner in order to be
able to control whether one, two, or three albumin binding residues (A)
should be incorporated in the growth hormone compound. The hydrophilic
spacer (B) may be attached to an amino acid side-chain of the GH
compound. Such amino acid side-chain may be a chemically modified amino
acid side-chain of the GH compound. Another, such amino acid side-chain
may be an enzymatically modified amino acid side-chain of the GH
compound. Preferably, a transglutaminase is used to introduce a
hydrophilic spacer in the glutamine residue in the position corresponding
to position 40 or 141 in SEQ ID No. 1. Another way of selectively
introducing a hydrophilic spacer is in the N-terminal residue of the
growth hormone compound, such as hGH (SEQ ID No. 1).

[0172] In the growth hormone conjugate of the formula (I) the fragment
A-W--B may be linear or branched. In one embodiment, A-W--B is not a
linear peptide.

[0173] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the position
corresponding to position 40 in SEQ ID No. 1.

[0174] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the position
corresponding to position 141 in SEQ ID No. 1.

[0175] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the N-terminal residue of the growth
hormone compound, such as hGH (SEQ ID No. 1).

[0176] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the position
corresponding to position 40 and to the glutamine residue in the position
corresponding to position 141 in SEQ ID No. 1.

[0177] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the position
corresponding to position 40 and to the N-terminal residue of the growth
hormone compound, such as hGH (SEQ ID No. 1).

[0178] In a further embodiment the albumin binding residue via a
hydrophilic spacer is attached to the glutamine residue in the position
corresponding to position 141 and to the N-terminal residue of the growth
hormone compound, such as hGH (SEQ ID No. 1).

[0179] The growth hormone conjugates of the present invention are selected
from

[0180] In a further aspect the present invention relates to a growth
hormone conjugate which comprises a growth hormone compound (GH) linked
to an albumin binding residue via a hydrophilic spacer, or a
pharmaceutically acceptable salt, solvate or prodrug thereof for use in
therapy. Furthermore, in the growth hormone conjugate of the present
invention GH, the albumin binding residue, and the hydrophilic spacer are
selected from any one of the above embodiments, in particular the growth
hormone conjugate has the formula (I) or (II).

[0181] In a further aspect the present invention relates to a
pharmaceutical composition comprising a growth hormone conjugate which
comprises a growth hormone compound (GH) linked to an albumin binding
residue via a hydrophilic spacer, or a pharmaceutically acceptable salt,
solvate or prodrug thereof, optionally in combination with a
pharmaceutical acceptable excipient.

[0182] The term "identity" as known in the art, refers to a relationship
between the sequences of two or more proteins, as determined by comparing
the sequences. In the art, "identity" also means the degree of sequence
relatedness between proteins, as determined by the number of matches
between strings of two or more amino acid residues. "Identity" measures
the percent of identical matches between the smaller of two or more
sequences with gap alignments (if any) addressed by a particular
mathematical model or computer program (i.e., "algorithms"). Identity of
related proteins can be readily calculated by known methods. Such methods
include, but are not limited to, those described in Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York,
1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part
1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J.
Applied Math., 48, 1073, (1988).

[0183] Preferred methods to determine identity are designed to give the
largest match between the sequences tested. Methods to determine identity
are described in publicly available computer programs. Preferred computer
program methods to determine identity between two sequences include the
GCG program package, including GAP (Devereux et al., Nucl. Acid. Res.,
12, 387, (1984); Genetics Computer Group, University of Wisconsin,
Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol.
Biol., 215, 403-410, (1990)). The BLASTX program is publicly available
from the National Center for Biotechnology Information (NCBI) and other
sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;
Altschul et al., supra). The well known Smith Waterman algorithm may also
be used to determine identity.

[0184] For example, using the computer algorithm GAP (Genetics Computer
Group, University of Wisconsin, Madison, Wis.), two proteins for which
the percent sequence identity is to be determined are aligned for optimal
matching of their respective amino acids (the "matched span", as
determined by the algorithm). A gap opening penalty (which is calculated
as 3× the average diagonal; the "average diagonal" is the average
of the diagonal of the comparison matrix being used; the "diagonal" is
the score or number assigned to each perfect amino acid match by the
particular comparison matrix) and a gap extension penalty (which is
usually 1/10 times the gap opening penalty), as well as a comparison
matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the
algorithm. A standard comparison matrix (see Dayhoff et al., Atlas of
Protein Sequence and Structure, vol. 5, supp.3 (1978) for the PAM 250
comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA, 89,
10915-10919, (1992) for the BLOSUM 62 comparison matrix) is also used by
the algorithm.

[0185] Preferred parameters for a protein sequence comparison include the
following:

[0187] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for protein
comparisons (along with no penalty for end gaps) using the GAP algorithm.

[0188] The compounds of the present invention have improved
pharmacological properties compared to the corresponding un-conjugated
growth hormone, which is also referred to as the parent compound.
Examples of such pharmacological properties include functional in vivo
half-life, immunogencity, renal filtration, protease protection and
albumin binding.

[0189] The term "functional in vivo half-life" is used in its normal
meaning, i.e., the time at which 50% of the biological activity of the GH
or GH conjugate is still present in the body/target organ, or the time at
which the activity of the GH or GH conjugate is 50% of its initial value.
As an alternative to determining functional in vivo half-life, "in vivo
plasma half-life" may be determined, i.e., the time at which 50% of the
GH or GH conjugate circulate in the plasma or bloodstream prior to being
cleared. Determination of plasma half-life is often more simple than
determining functional half-life and the magnitude of plasma half-life is
usually a good indication of the magnitude of functional in vivo
half-life. Alternative terms to plasma half-life include serum half-life,
circulating half-life, circulatory half-life, serum clearance, plasma
clearance, and clearance half-life.

[0190] The term "increased" as used in connection with the functional in
vivo half-life or plasma half-life is used to indicate that the relevant
half-life of the GH conjugate is statistically significantly increased
relative to that of the parent GH, as determined under comparable
conditions. For instance the relevant half-life may be increased by at
least about 25%, such as by at lest about 50%, e.g., by at least about
100%, 150%, 200%, 250%, or 500%. In one embodiment, the compounds of the
present invention exhibit an increase in half-life of at least about 5 h,
preferably at least about 24 h, more preferably at least about 72 h, and
most preferably at least about 7 days, relative to the half-life of the
parent GH.

[0191] Measurement of in vivo plasma half-life can be carried out in a
number of ways as described in the literature. An increase in in vivo
plasma half-life may be quantified as a decrease in clearance (CL) or as
an increase in mean residence time (MRT). Conjugated GH of the present
invention for which the CL is decreased to less than 70%, such as less
than 50%, such than less than 20%, such than less than 10% of the CL of
the parent GH as determined in a suitable assay is said to have an
increased in vivo plasma half-life. Conjugated GH of the present
invention for which MRT is increased to more than 130%, such as more than
150%, such as more than 200%, such as more than 500% of the MRT of the
parent GH in a suitable assay is said to have an increased in vivo plasma
half-life. Clearance and mean residence time can be assessed in standard
pharmacokinetic studies using suitable test animals. It is within the
capabilities of a person skilled in the art to choose a suitable test
animal for a given protein. Tests in human, of course, represent the
ultimate test. Suitable text animals include normal, Sprague-Dawley male
rats, mice and cynomolgus monkeys. Typically the mice and rats are in
injected in a single subcutaneous bolus, while monkeys may be injected in
a single subcutaneous bolus or in a single iv dose. The amount injected
depends on the test animal. Subsequently, blood samples are taken over a
period of one to five days as appropriate for the assessment of CL and
MRT. The blood samples are conveniently analysed by ELISA techniques.

[0192] The term "Immunogenicity" of a compound refers to the ability of
the compound, when administered to a human, to elicit a deleterious
immune response, whether humoral, cellular, or both. In any human
sub-population, there may be individuals who exhibit sensitivity to
particular administered proteins. Immunogenicity may be measured by
quantifying the presence of growth hormone antibodies and/or growth
hormone responsive T-cells in a sensitive individual, using conventional
methods known in the art. In one embodiment, the conjugated GH of the
present invention exhibit a decrease in immunogenicity in a sensitive
individual of at least about 10%, preferably at least about 25%, more
preferably at least about 40% and most preferably at least about 50%,
relative to the immunogenicity for that individual of the parent GH.

[0193] The term "protease protection" or "protease protected" as used
herein is intended to indicate that the conjugated GH of the present
invention is more resistant to the plasma peptidase or proteases than is
the parent GH. Protease and peptidase enzymes present in plasma are known
to be involved in the degradation of circulating proteins, such as e.g.
circulating peptide hormones, such as growth hormone.

[0194] Growth hormone may be susceptible to degradation by for instance
thrombin, plasmin, subtilisin, and chymotrypsin-like serine proteinase.
Assays for determination of degradation of these proteases are described
in J. Biotech., 65, 183, (1998). In one embodiment, the rate of
hydrolysis of the GH conjugate is less than 70%, such as less than 40%,
such as less than 10% of that of the parent GH.

[0195] The most abundant protein component in circulating blood of
mammalian species is serum albumin, which is normally present at a
concentration of approximately 3 to 4.5 grams per 100 milliliters of
whole blood. Serum albumin is a blood protein of approximately 70,000
daltons which has several important functions in the circulatory system.
It functions as a transporter of a variety of organic molecules found in
the blood, as the main transporter of various metabolites such as fatty
acids and bilirubin through the blood, and, owing to its abundance, as an
osmotic regulator of the circulating blood. Serum albumin has a half-life
of more than one week, and one approach to increasing the plasma
half-life of proteins has been to conjugate to the protein a group that
binds to serum albumin. Albumin binding property may be determined as
described in J. Med. Chem., 43, 1986, (2000) which is incorporated herein
by reference.

[0196] The growth hormone conjugates of formula (I) or (II) exert growth
hormone activity and may as such be used in the treatment of diseases or
states which will benefit from an increase in the amount of circulating
growth hormone. In particular, the invention provides a method for the
treatment of growth hormone deficiency (GHD); Turner Syndrome;
Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome; chronic
renal disease, juvenile rheumatoid arthritis; cystic fibrosis,
HIV-infection in children receiving HAART treatment (HIV/HALS children);
short children born short for gestational age (SGA); short stature in
children born with very low birth weight (VLBW) but SGA; skeletal
dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature
(ISS); GHD in adults; fractures in or of long bones, such as tibia,
fibula, femur, humerus, radius, ulna, clavicula, matacarpea, matatarsea,
and digit; fractures in or of spongious bones, such as the scull, base of
hand, and base of food; patients after tendon or ligament surgery in e.g.
hand, knee, or shoulder; patients having or going through distraction
osteogenesis; patients after hip or discus replacement, meniscus repair,
spinal fusions or prosthesis fixation, such as in the knee, hip,
shoulder, elbow, wrist or jaw; patients into which osteosynthesis
material, such as nails, screws and plates, have been fixed; patients
with non-union or mal-union of fractures; patients after osteatomia, e.g.
from tibia or 1st toe; patients after graft implantation; articular
cartilage degeneration in knee caused by trauma or arthritis;
osteoporosis in patients with Turner syndrome; osteoporosis in men; adult
patients in chronic dialysis (APCD); malnutritional associated
cardiovascular disease in APCD; reversal of cachexia in APCD; cancer in
APCD; chronic abstractive pulmonal disease in APCD; HIV in APCD; elderly
with APCD; chronic liver disease in APCD, fatigue syndrome in APCD;
Crohn's disease; impaired liver function; males with HIV infections;
short bowel syndrome; central obesity; HIV-associated lipodystrophy
syndrome (HALS); male infertility; patients after major elective surgery,
alcohol/drug detoxification or neurological trauma; aging; frail elderly;
osteo-arthritis; traumatically damaged cartilage; erectile dysfunction;
fibromyalgia; memory disorders; depression; traumatic brain injury;
subarachnoid haemorrhage; very low birth weight; metabolic syndrome;
glucocorticoid myopathy; or short stature due to glucucorticoid treatment
in children, the method comprising administering to a patient in need
thereof a therapeutically effective amount of a growth hormone conjugate
according to formula (I) or (II).

[0197] In a further aspect, the invention provides a method for the
acceleration of the healing of muscle tissue, nervous tissue or wounds;
the acceleration or improvement of blood flow to damaged tissue; or the
decrease of infection rate in damaged tissue, the method comprising
administration to a patient in need thereof an effective amount of a
therapeutically effective amount of a growth hormone conjugate of formula
(I) or (II).

[0198] In a further embodiment, the invention relates to the use of a
growth hormone conjugate of formula (I) or (II) in the manufacture of
diseases benefiting from an increase in the growth hormone plasma level,
such as the disease mentioned above.

[0199] A typical parenteral dose is in the range of 10-9 mg/kg to
about 100 mg/kg body weight per administration. Typical administration
doses are from about 0.0000001 to about 10 mg/kg body weight per
administration. The exact dose will depend on e.g. indication,
medicament, frequency and mode of administration, the sex, age and
general condition of the subject to be treated, the nature and the
severity of the disease or condition to be treated, the desired effect of
the treatment and other factors evident to the person skilled in the art.

[0200] Typical dosing frequencies are twice daily, once daily, bi-daily,
twice weekly, once weekly or with even longer dosing intervals. Due to
the prolonged half-lives of the fusion proteins of the present invention,
a dosing regime with long dosing intervals, such as twice weekly, once
weekly or with even longer dosing intervals is a particular embodiment of
the invention.

[0201] Many diseases are treated using more than one medicament in the
treatment, either concomitantly administered or sequentially
administered. It is therefore within the scope of the present invention
to use a growth hormone conjugate of formula (I) or (II) in therapeutic
methods for the treatment of one of the above mentioned diseases in
combination with one or more other therapeutically active compound(s)
normally used in the treatment said diseases. By analogy, it is also
within the scope of the present invention to use a growth hormone
conjugate of formula (I) or (II)in combination with other therapeutically
active compounds normally used in the treatment of one of the above
mentioned diseases in the manufacture of a medicament for said disease.

[0202] General Methods of Preparation

Enzyme Conjugation:

[0203] In the preparation of a growth hormone conjugate of the present
invention, typically at least one of the covalent bonds established in
the preparation of a A-W--B-GH conjugate of formula (I) is prepared by
use of an enzyme as illustrated in the examples below. Such an enzyme may
for instance be selected from the group consisting of transglutaminases,
serine proteases and cysteine proteases. Typically, said enzyme is a
transglutaminase. Such transglutaminase may for instance be selected from
the group consisting of microbial transglutaminases, tissue
transglutaminases and factor XIII and variants thereof. In another
embodiment, said enzyme is a cysteine protease. Such a cysteine protease
may for instance be selected from the group consisting of papain, sortase
A and sortase B. In a further embodiment, said enzyme is a serine
protease. Such a serine protease may for instance be selected from the
group consisting of carboxypeptidase Y (CPY) (PCT application
WO2005/035553 contains general disclosure of protein modification using
CPY), trypsin and chymotrypsin.

[0204] The growth hormone conjugate of the present invention may be
prepared by many different methods, non-limiting examples are shown
below.

[0206] As stated above, at least one of the covalent bonds established in
the preparation of a A-W--B-GH conjugate of the present invention may be
prepared by use of a transglutaminase. Transglutaminases may include
microbial transglutaminases such as that isolated from the Streptomyces
species; S. mobaraense, S. cinnamoneum, S. griseocarneum (U.S. Pat. No.
5,156,956 incorporated herein by reference), S. lavendulae (U.S. Pat. No.
5,252,469 incorporated herein by reference) and Streptomyces ladakanum
(JP2003/199569 incorporated herein by reference). Other useful microbial
transglutaminases have been isolated from Bacillus subtilis (disclosed in
U.S. Pat. No. 5,731,183, which is incorporated herein by reference) and
from various Myxomycetes. Other examples of useful microbial
transglutaminases are those disclosed in WO96/06931 (e.g.
transglutaminase from Bacillus lydicus) and WO96/22366, both of which are
incorporated herein by reference. Useful non-microbial transglutaminases
include guinea-pig liver transglutaminase, and transglutaminases from
various marine sources like the flat fish Pagrus major (disclosed in
EP0555649, which is incorporated herein by reference), and the Japanese
oyster Crassostrea gigas (disclosed in U.S. Pat. No. 5,736,356, which is
incorporated herein by reference). Functional analogues and derivatives
thereof may also be useful.

[0207] Typically, the TGase used in the methods of the invention is a
microbial transglutaminase. In one embodiment, the TGase is from S.
mobaraense or a variant thereof, for instance as described in
WO2007/020290 and WO2008/020075. In another embodiment, the TGase is from
S. ladakanum or a variant thereof, for instance as described in
WO2008/020075.

[0208] The conjugation of hGH to A-W--B according to the present invention
may be achieved by TGase-mediated modification leading to selective
alteration at specific lysine (Lys) or glutamine (Gln) positions in the
sequence of the GH compound depending on the substrate used. Use of
amines as substrates will lead to modification of Glutamines whereas the
use of primary amides will lead to modification of Lysines. hGH (SEQ ID
No. 1) has 9 lysine residues at positions 38, 41, 70, 115, 140, 145, 158,
168 and 172 and 13 glutamine residues at positions 22, 29, 40, 46, 49,
68, 69, 84, 91, 122, 137, 141 and 181. Not all of these are readily
available for modification nor suitably for modifications as this will
lead to diminished binding potency to the growth hormone binding protein
hence leading to reduced biological activity. The x-ray protein crystal
structure between hGH and its binding protein (pdb: 3HHR) reveals that at
least 4 lysines (38, 41, 168 and 172) takes part in binding to the
binding protein and potentially only one of the glutamines (Gln 46). This
render the glutamines more attractive as target for selective
introduction of an albumin binder linker. These structural considerations
are further supported by findings summarised by N. Ch ne et al in Reprod.
Nutr. Develop. 29, 1-25 (1989) where it's concluded that chemical
modifications affecting lysines have been found to have a negative effect
on the in vivo biological activity and on the binding capacity to the
liver receptors of GH.

Chemistry I

[0209] In an aspect the present invention relates to preparation of a
growth hormone conjugate of formula (VI) wherein a GH compound is treated
with a property-modifying group-derived aldehyde or ketone to yield an
amine, imine or a hemiaminal.

[0210] In a further aspect the present invention relates to preparation of
a growth hormone conjugate of formula (VI) comprising treatment of an
aldehyde or ketone derived from the GH compound with a property-modifying
group-derived aniline or heteroarylamine to yield an amine, imine or a
hemiaminal.

[0211] In an embodiment, aldehyde derived from the GH compound (IV) is
treated with property-modifying group-derived aniline or heteroarylamine
(V).

[0212] The term "GH compound derived aldehyde (or ketone)" or "an aldehyde
or ketone derived from the GH compound" is intended to indicate a GH
compound to which an aldehyde or ketone functional group has been
covalently attached, or a GH compound on which an aldehyde or ketone
functional group has been generated. The preparation of GH
compound-derived aldehydes, such as compound (IV) illustrated below is
well known to those skilled in the art, and any of these known procedures
may be used to prepare the GH compound-derived aldehyde (IV) required for
the realization of the invention disclosed herein.

[0213] In one embodiment, the conjugate A-W--B-hGH (VI) is prepared as
illustrated below:

##STR00016##

[0214] The TGase-mediated enzymatic reaction with GH (I) results in the
modification of Gln at position 141 and/or 40 affording GH (II).
Conjugation of periodate oxidised GH (II) with A-W--B-NH2 (V) occurs via
reductive alkylation. Reductive alkylation is exemplified herein and is
well-recognized in the art and results in GH compounds (VI) modified in
position Gln(141) and/or 40.

Chemistry II

[0215] In one embodiment, the conjugate A-W--B-hGH is prepared as
illustrated below:

##STR00017##

[0216] The derivatization process as shown above provides an albumin
binding linker attached to hGH compound in position Phe(1).

[0217] A close relationship to the natural peptide is generally regarded
as an advantage with therapeutic interventions comprising administration
of variants or analogues of this natural peptide as it minimizes the risk
of e.g. any unwanted antibody generation.

[0218] Many nucleophilic compounds are known which could be incorporated
into peptides according to the methods of the present invention, and
a-amino acids are one such type of nucleophilic compounds. For the
purpose of the present invention, it is, however, preferred to select the
nucleophilic compound so that the transacylated compound formed is not
itself a substrate for the enzyme applied. Stated differently, it is
preferred to apply a nucleophilic compound which effectively blocks any
further reaction of the enzyme. One example of such compounds is amides
of a-amino acids as carboxy amidated peptides are not substrates for
carboxypeptidases.

[0219] It is recognised that whether or not a compound is a substrate for
a given enzyme in principle depends on the conditions, e.g. the time
frame, under which the reaction takes place. Given sufficient time, many
compounds are, in fact, substrates for an enzyme although they are not
under normal conditions regarded as such. When it is stated above that
the transacylated compound itself should not be a substrate of the enzyme
it is intended to indicate that the transacylated compound itself is not
a substrate for the enzyme to an extent where the following reactions in
the method of the present invention are disturbed. If the transacylated
compound is, in fact, a substrate for the enzyme, the enzyme may be
removed or inactivated, e.g. by enzyme inhibitors, following the
transacylation reaction.

Pharmaceutical Compositions

[0220] Another purpose is to provide a pharmaceutical composition
comprising a growth hormone conjugate of the present invention, such as a
growth hormone conjugate of formula (I) or (II), which is present in a
concentration from 10-15 mg/mL to 200 mg/mL, such as e.g. 10-10
mg/mL to 5 mg/mL and wherein said composition has a pH from 2.0 to 10.0.
The composition may further comprise pharmaceutical exhibits, such as a
buffer system, preservative(s), tonicity agent(s), chelating agent(s),
stabilizers and surfactants. In one embodiment of the invention the
pharmaceutical composition is an aqueous composition, i.e. composition
comprising water. Such composition is typically a solution or a
suspension. In a further embodiment of the invention the pharmaceutical
composition is an aqueous solution. The term "aqueous composition" is
defined as a composition comprising at least 50% w/w water. Likewise, the
term "aqueous solution" is defined as a solution comprising at least 50%
w/w water, and the term "aqueous suspension" is defined as a suspension
comprising at least 50% w/w water.

[0221] In another embodiment the pharmaceutical composition is a
freeze-dried composition, whereto the physician or the patient adds
solvents and/or diluents prior to use.

[0222] In another embodiment the pharmaceutical composition is a dried
composition (e.g. freeze-dried or spray-dried) ready for use without any
prior dissolution.

[0223] In a further aspect the invention relates to a pharmaceutical
composition comprising an aqueous solution of a growth hormone conjugate,
such as a growth hormone conjugate of formula (I) or (II), and a buffer,
wherein said GH conjugate is present in a concentration from 0.1-100
mg/mL or above, and wherein said composition has a pH from about 2.0 to
about 10.0.

[0225] In a further embodiment of the invention the buffer is selected
from the group consisting of sodium acetate, sodium carbonate, citrate,
glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen
phosphate, disodium hydrogen phosphate, sodium phosphate, and
tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,
maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures
thereof. Each one of these specific buffers constitutes an alternative
embodiment of the invention.

[0226] In a further embodiment of the invention the composition further
comprises a pharmaceutically acceptable preservative. In a further
embodiment of the invention the preservative is selected from the group
consisting of phenol, o-cresol, m-cresol, p-cresol, methyl
p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl
p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and
thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium
dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium
chloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixtures
thereof. In a further embodiment of the invention the preservative is
present in a concentration from 0.1 mg/mL to 20 mg/mL. In a further
embodiment of the invention the preservative is present in a
concentration from 0.1 mg/mL to 5 mg/mL. In a further embodiment of the
invention the preservative is present in a concentration from 5 mg/mL to
10 mg/mL. In a further embodiment of the invention the preservative is
present in a concentration from 10 mg/mL to 20 mg/mL. Each one of these
specific preservatives constitutes an alternative embodiment of the
invention. The use of a preservative in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made to
Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

[0227] In a further embodiment of the invention the composition further
comprises an isotonic agent. In a further embodiment of the invention the
isotonic agent is selected from the group consisting of a salt (e.g.
sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.
L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,
tryptophan, threonine), an alditol (e.g. glycerol (glycerine),
1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)
polyethyleneglycol (e.g. PEG 400), or mixtures thereof. Any sugar such as
mono-, di-, or polysaccharides, or water-soluble glucans, including for
example fructose, glucose, mannose, sorbose, xylose, maltose, lactose,
sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble
starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In
one embodiment the sugar additive is sucrose. Sugar alcohol is defined as
a C4-C8 hydrocarbon having at least one --OH group and includes, for
example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and
arabitol. In one embodiment the sugar alcohol additive is mannitol. The
sugars or sugar alcohols mentioned above may be used individually or in
combination. There is no fixed limit to the amount used, as long as the
sugar or sugar alcohol is soluble in the liquid preparation and does not
adversely effect the stabilizing effects obtained using the methods of
the invention. In one embodiment, the sugar or sugar alcohol
concentration is between about 1 mg/mL and about 150 mg/mL. In a further
embodiment of the invention the isotonic agent is present in a
concentration from 1 mg/mL to 50 mg/mL. In a further embodiment of the
invention the isotonic agent is present in a concentration from 1 mg/mL
to 7 mg/mL. In a further embodiment of the invention the isotonic agent
is present in a concentration from 8 mg/mL to 24 mg/mL. In a further
embodiment of the invention the isotonic agent is present in a
concentration from 25 mg/mL to 50 mg/mL. Each one of these specific
isotonic agents constitutes an alternative embodiment of the invention.
The use of an isotonic agent in pharmaceutical compositions is well-known
to the skilled person. For convenience reference is made to Remington:
The Science and Practice of Pharmacy, 20th edition, 2000.

[0228] In a further embodiment of the invention the composition further
comprises a chelating agent. In a further embodiment of the invention the
chelating agent is selected from salts of ethylenediaminetetraacetic acid
(EDTA), citric acid, and aspartic acid, and mixtures thereof. In a
further embodiment of the invention the chelating agent is present in a
concentration from 0.1 mg/mL to 5 mg/mL. In a further embodiment of the
invention the chelating agent is present in a concentration from 0.1
mg/mL to 2 mg/mL. In a further embodiment of the invention the chelating
agent is present in a concentration from 2 mg/mL to 5 mg/mL. Each one of
these specific chelating agents constitutes an alternative embodiment of
the invention. The use of a chelating agent in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of Pharmacy,
20th edition, 2000.

[0229] In a further embodiment of the invention the composition further
comprises a stabilizer. The use of a stabilizer in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of Pharmacy,
20th edition, 2000.

[0230] More particularly, compositions of the invention are stabilized
liquid pharmaceutical compositions whose therapeutically active
components include a protein that possibly exhibits aggregate formation
during storage in liquid pharmaceutical compositions. By "aggregate
formation" is intended a physical interaction between the protein
molecules that results in formation of oligomers, which may remain
soluble, or large visible aggregates that precipitate from the solution.
By "during storage" is intended a liquid pharmaceutical composition or
composition once prepared, is not immediately administered to a subject.
Rather, following preparation, it is packaged for storage, either in a
liquid form, in a frozen state, or in a dried form for later
reconstitution into a liquid form or other form suitable for
administration to a subject. By "dried form" is intended the liquid
pharmaceutical composition or composition is dried either by freeze
drying (i.e., lyophilization; see, for example, Williams and Polli, J.
Parenteral Sci. Technol. 38, 48-59, (1984)), spray drying (see Masters
(1991) in Spray-Drying Handbook (5th ed; Longman Scientific and
Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel.
Ind. Pharm. 18, 1169-1206, (1992); and Mumenthaler et al., Pharm. Res.
11, 12-20 (1994)), or air drying (Carpenter and Crowe, Cryobiology 25,
459-470, (1988); and Roser, Biopharm. 4, 47-53, (1991)). Aggregate
formation by a protein during storage of a liquid pharmaceutical
composition can adversely affect biological activity of that protein,
resulting in loss of therapeutic efficacy of the pharmaceutical
composition. Furthermore, aggregate formation may cause other problems
such as blockage of tubing, membranes, or pumps when the
protein-containing pharmaceutical composition is administered using an
infusion system.

[0231] The pharmaceutical compositions of the invention may further
comprise an amount of an amino acid base sufficient to decrease aggregate
formation by the protein during storage of the composition. By "amino
acid base" is intended an amino acid or a combination of amino acids,
where any given amino acid is present either in its free base form or in
its salt form. Where a combination of amino acids is used, all of the
amino acids may be present in their free base forms, all may be present
in their salt forms, or some may be present in their free base forms
while others are present in their salt forms. In one embodiment, amino
acids to use in preparing the compositions of the invention are those
carrying a charged side chain, such as arginine, lysine, aspartic acid,
and glutamic acid. Any stereoisomer (i.e., L or D isomer, or mixtures
thereof) of a particular amino acid (methionine, histidine, arginine,
lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures
thereof) or combinations of these stereoisomers or glycine or an organic
base such as but not limited to imidazole, may be present in the
pharmaceutical compositions of the invention so long as the particular
amino acid or organic base is present either in its free base form or its
salt form. In one embodiment the L-stereoisomer of an amino acid is used.
In one embodiment the L-stereoisomer is used. Compositions of the
invention may also be formulated with analogues of these amino acids. By
"amino acid analogue" is intended a derivative of the naturally occurring
amino acid that brings about the desired effect of decreasing aggregate
formation by the protein during storage of the liquid pharmaceutical
compositions of the invention. Suitable arginine analogues include, for
example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable
methionine analogues include ethionine and buthionine and suitable
cysteine analogues include S-methyl-L cysteine. As with the other amino
acids, the amino acid analogues are incorporated into the compositions in
either their free base form or their salt form. In a further embodiment
of the invention the amino acids or amino acid analogues are used in a
concentration, which is sufficient to prevent or delay aggregation of the
protein.

[0232] In a further embodiment of the invention methionine (or other
sulphuric amino acids or amino acid analogous) may be added to inhibit
oxidation of methionine residues to methionine sulfoxide when the protein
acting as the therapeutic agent is a protein comprising at least one
methionine residue susceptible to such oxidation. By "inhibit" is
intended minimal accumulation of methionine oxidized species over time.
Inhibiting methionine oxidation results in greater retention of the
protein in its proper molecular form. Any stereoisomer of methionine (L
or D isomer) or any combinations thereof can be used. The amount to be
added should be an amount sufficient to inhibit oxidation of the
methionine residues such that the amount of methionine sulfoxide is
acceptable to regulatory agencies. Typically, this means that the
composition contains no more than about 10% to about 30% methionine
sulfoxide. Generally, this can be obtained by adding methionine such that
the ratio of methionine added to methionine residues ranges from about
1:1 to about 1000:1, such as 10:1 to about 100:1.

[0233] In a further embodiment of the invention the composition further
comprises a stabilizer selected from the group of high molecular weight
polymers or low molecular compounds. In a further embodiment of the
invention the stabilizer is selected from polyethylene glycol (e.g. PEG
3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,
carboxy-/hydroxycellulose or derivatives thereof (e.g. HPC, HPC-SL, HPC-L
and HPMC), cyclodextrins, sulphur-containing substances as
monothioglycerol, thioglycolic acid and 2-methylthioethanol, and
different salts (e.g. sodium chloride). Each one of these specific
stabilizers constitutes an alternative embodiment of the invention.

[0234] The pharmaceutical compositions may also comprise additional
stabilizing agents, which further enhance stability of a therapeutically
active protein therein. Stabilizing agents of particular interest to the
present invention include, but are not limited to, methionine and EDTA,
which protect the protein against methionine oxidation, and a nonionic
surfactant, which protects the protein against aggregation associated
with freeze-thawing or mechanical shearing.

[0236] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made to
Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

[0237] It is possible that other ingredients may be present in the
pharmaceutical composition of the present invention. Such additional
ingredients may include wetting agents, emulsifiers, antioxidants,
bulking agents, tonicity modifiers, chelating agents, metal ions,
oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or
proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine,
arginine, glycine, lysine and histidine). Such additional ingredients, of
course, should not adversely affect the overall stability of the
pharmaceutical composition of the present invention.

[0238] Pharmaceutical compositions containing a growth hormone conjugate
according to the present invention may be administered to a patient in
need of such treatment at several sites, for example, at topical sites,
for example, skin and mucosal sites, at sites which bypass absorption,
for example, administration in an artery, in a vein, in the heart, and at
sites which involve absorption, for example, administration in the skin,
under the skin, in a muscle or in the abdomen.

[0239] Administration of pharmaceutical compositions according to the
invention may be through several routes of administration, for example,
lingual, sublingual, buccal, in the mouth, oral, in the stomach and
intestine, nasal, pulmonary, for example, through the bronchioles and
alveoli or a combination thereof, epidermal, dermal, transdermal,
vaginal, rectal, ocular, for examples through the conjunctiva, urethral,
and parenteral to patients in need of such a treatment.

[0241] Compositions of the invention may further be compounded in, or
attached to, for example through covalent, hydrophobic and electrostatic
interactions, a drug carrier, drug-delivery system and advanced drug
delivery system in order to further enhance stability of the growth
hormone conjugate, increase bioavailability, increase solubility,
decrease adverse effects, achieve chronotherapy well known to those
skilled in the art, and increase patient compliance or any combination
thereof. Examples of carriers, drug delivery systems and advanced drug
delivery systems include, but are not limited to, polymers, for example
cellulose and derivatives, polysaccharides, for example dextran and
derivatives, starch and derivatives, poly(vinyl alcohol), acrylate and
methacrylate polymers, polylactic and polyglycolic acid and block
co-polymers thereof, polyethylene glycols, carrier proteins, for example
albumin, gels, for example, thermogelling systems, for example block
co-polymeric systems well known to those skilled in the art, micelles,
liposomes, microspheres, nanoparticulates, liquid crystals and
dispersions thereof, L2 phase and dispersions there of, well known to
those skilled in the art of phase behaviour in lipid-water systems,
polymeric micelles, multiple emulsions, self-emulsifying,
self-microemulsifying, cyclodextrins and derivatives thereof, and
dendrimers.

[0242] Compositions of the current invention are useful in the composition
of solids, semi-solids, powder and solutions for pulmonary administration
of growth hormone conjugate, using, for example a metered dose inhaler,
dry powder inhaler and a nebulizer, all being devices well known to those
skilled in the art.

[0243] Compositions of the current invention are specifically useful in
the composition of controlled, sustained, protracting, retarded, and slow
release drug delivery systems. More specifically, but not limited to,
compositions are useful in composition of parenteral controlled release
and sustained release systems (both systems leading to a many-fold
reduction in number of administrations), well known to those skilled in
the art. Even more preferably, are controlled release and sustained
release systems administered subcutaneous. Without limiting the scope of
the invention, examples of useful controlled release system and
compositions are hydrogels, oleaginous gels, liquid crystals, polymeric
micelles, microspheres, nanoparticles,

[0245] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of a
syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A further
option is a composition which may be a solution or suspension for the
administration of the growth hormone conjugate in the form of a nasal or
pulmonal spray. As a still further option, the pharmaceutical
compositions containing the growth hormone conjugate of the invention can
also be adapted to transdermal administration, e.g. by needle-free
injection or from a patch, optionally an iontophoretic patch, or
transmucosal, e.g. buccal, administration.

[0246] The term "stabilized composition" refers to a composition with
increased physical stability, increased chemical stability or increased
physical and chemical stability.

[0247] The term "physical stability" of the protein composition as used
herein refers to the tendency of the protein to form biologically
inactive and/or insoluble aggregates of the protein as a result of
exposure of the protein to thermo-mechanical stresses and/or interaction
with interfaces and surfaces that are destabilizing, such as hydrophobic
surfaces and interfaces. Physical stability of the aqueous protein
compositions is evaluated by means of visual inspection and/or turbidity
measurements after exposing the composition filled in suitable containers
(e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual inspection of
the compositions is performed in a sharp focused light with a dark
background. The turbidity of the composition is characterized by a visual
score ranking the degree of turbidity for instance on a scale from 0 to 3
(a composition showing no turbidity corresponds to a visual score 0, and
a composition showing visual turbidity in daylight corresponds to visual
score 3). A composition is classified physical unstable with respect to
protein aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the composition can be evaluated by
simple turbidity measurements well-known to the skilled person. Physical
stability of the aqueous protein compositions can also be evaluated by
using a spectroscopic agent or probe of the conformational status of the
protein. The probe is preferably a small molecule that preferentially
binds to a non-native conformer of the protein. One example of a small
molecular spectroscopic probe of protein structure is Thioflavin T.
Thioflavin T is a fluorescent dye that has been widely used for the
detection of amyloid fibrils. In the presence of fibrils, and perhaps
other protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about 482 nm
when bound to a fibril protein form. Unbound Thioflavin T is essentially
non-fluorescent at the wavelengths.

[0248] Other small molecules can be used as probes of the changes in
protein structure from native to non-native states. For instance the
"hydrophobic patch" probes that bind preferentially to exposed
hydrophobic patches of a protein. The hydrophobic patches are generally
buried within the tertiary structure of a protein in its native state,
but become exposed as a protein begins to unfold or denature. Examples of
these small molecular, spectroscopic probes are aromatic, hydrophobic
dyes, such as anthracene, acridine, phenanthroline or the like. Other
spectroscopic probes are metal-amino acid complexes, such as cobalt metal
complexes of hydrophobic amino acids, such as phenylalanine, leucine,
isoleucine, methionine, and valine, or the like.

[0249] The term "chemical stability" of the protein composition as used
herein refers to chemical covalent changes in the protein structure
leading to formation of chemical degradation products with potential less
biological potency and/or potential increased immunogenic properties
compared to the native protein structure. Various chemical degradation
products can be formed depending on the type and nature of the native
protein and the environment to which the protein is exposed. Elimination
of chemical degradation can most probably not be completely avoided and
increasing amounts of chemical degradation products is often seen during
storage and use of the protein composition as well-known by the person
skilled in the art. Most proteins are prone to deamidation, a process in
which the side chain amide group in glutaminyl or asparaginyl residues is
hydrolysed to form a free carboxylic acid. Other degradations pathways
involves formation of high molecular weight transformation products where
two or more protein molecules are covalently bound to each other through
transamidation and/or disulfide interactions leading to formation of
covalently bound dimer, oligomer and polymer degradation products
(Stability of Protein Pharmaceuticals, Ahern. T. J. & Manning M. C.,
Plenum Press, New York 1992). Oxidation (of for instance methionine
residues) can be mentioned as another variant of chemical degradation.
The chemical stability of the protein composition can be evaluated by
measuring the amount of the chemical degradation products at various
time-points after exposure to different environmental conditions (the
formation of degradation products can often be accelerated by for
instance increasing temperature). The amount of each individual
degradation product is often determined by separation of the degradation
products depending on molecule size and/or charge using various
chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

[0250] Hence, as outlined above, a "stabilized composition" refers to a
composition with increased physical stability, increased chemical
stability or increased physical and chemical stability. In general, a
composition must be stable during use and storage (in compliance with
recommended use and storage conditions) until the expiration date is
reached.

[0251] In one embodiment of the invention the pharmaceutical composition
comprising the growth hormone conjugate of formula (I) or (II)is stable
for more than 6 weeks of usage and for more than 3 years of storage.

[0252] In another embodiment of the invention the pharmaceutical
composition comprising the growth hormone conjugate of formula (I) or
(II)is stable for more than 4 weeks of usage and for more than 3 years of
storage.

[0253] In a further embodiment of the invention the pharmaceutical
composition comprising the growth hormone conjugate of formula (I) or
(II) is stable for more than 4 weeks of usage and for more than two years
of storage.

[0254] In an even further embodiment of the invention the pharmaceutical
composition comprising the growth hormone conjugate of formula (I) or
(II) is stable for more than 2 weeks of usage and for more than two years
of storage.

[0255] All references, including publications, patent applications and
patents, cited herein are hereby incorporated by reference to the same
extent as if each reference was individually and specifically indicated
to be incorporated by reference and was set forth in its entirety herein.

[0256] All headings and sub-headings are used herein for convenience only
and should not be construed as limiting the invention in any way,

[0257] Any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.

[0258] The terms "a" and "an" and "the" and similar referents as used in
the context of describing the invention are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or clearly
contradicted by context.

[0259] Recitation of ranges of values herein are merely intended to serve
as a shorthand method of referring individually to each separate value
falling within the range, unless otherwise indicated herein, and each
separate value is incorporated into the specification as if it were
individually recited herein. Unless otherwise stated, all exact values
provided herein are representative of corresponding approximate values
(e.g., all exact exemplary values provided with respect to a particular
factor or measurement can be considered to also pro-vide a corresponding
approximate measurement, modified by "about," where appropriate).

[0260] All methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted by
context.

[0261] The use of any and all examples, or exemplary language (e.g., "such
as") provided herein, is intended merely to better illuminate the
invention and does not pose a limitation on the scope of the invention
unless otherwise indicated. No language in the specification should be
construed as indicating any element is essential to the practice of the
invention unless as much is explicitly stated.

[0262] The citation and incorporation of patent documents herein is done
for convenience only and does not reflect any view of the validity,
patentability and/or enforceability of such patent documents.

[0263] The invention is further described by, but not limited to, the
embodiments listed here below. [0264] 1. A growth hormone conjugate which
comprises a growth hormone compound (GH) linked to an albumin binding
residue via a hydrophilic spacer, or a pharmaceutically acceptable salt,
solvate or prodrug thereof. [0265] 2. The conjugate of embodiment 1
wherein the hydrophilic spacer has mLogP<0 or a cLogP<0.5. [0266]
3. The conjugate of embodiment 1 or 2 wherein the growth hormone compound
(GH) is linked to one albumin binding residue via a hydrophilic spacer.
[0267] 4. The conjugate of embodiment 1 or 2 wherein the growth hormone
compound (GH) is linked to two or three albumin binding residues via a
hydrophilic spacer. [0268] 5. The conjugate of any one of embodiments 1-4
wherein the hydrophilic spacer has the formula

wherein [0302] Y is
--(CH2)17--C3-10-Cycloalkyl-W8-- or a valence bond,
[0303] I7 is 0-6, [0304] W7 is selected from --C(O)NH--, --NHC(O)--,
--C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH2--,
--CH2C(O)--, --C(O)CH═CH--, --CH═CHC(O)--,
--(CH2).sub.s3--, --C(O)--, --C(O)O--, --OC(O)--, or a valence bond;
wherein s3 is 0 or 1, [0305] W8 is selected from --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)--, --OC(O)NH--, --NHC(O)O--, --C(O)CH2--,
--CH2C(O)--, --C(O)CH═CH--, --CH═CHC(O)--,
--(CH2).sub.s4--, --C(O)--, --C(O)O--, --OC(O)--, or a valence bond;
wherein s4 is 0 or 1. [0306] 11. The conjugate of any one of
embodiments 6-10, wherein I1, I2, I3, I4, I5 and I6 independently are
0-6. [0307] 12. The conjugate of any one of embodiments 6-11, wherein m1,
m3, m4, m6 and m7 independently are 0-6. [0308] 13. The conjugate of any
one of embodiments 6-12, wherein m2 and m5 independently are 0-10. [0309]
14. The conjugate of any one of embodiments 6-13, wherein n1, n2, n3 and
n4 independently are 0-10, such as 0-6. [0310] 15. The conjugate of any
one of embodiments 6-14, wherein D1 and D2 are independently selected
from --O-- or --NR6-- or a valence bond. [0311] 16. The conjugate of
any one of embodiments 6-15, wherein D1 is --NR6--. [0312] 17. The
conjugate of any one of embodiments 6-16, wherein D2 is --NR6--.
[0313] 18. The conjugate of any one of embodiments 6-17, wherein D1 and
D2 are both --O--. [0314] 19. The conjugate of any one of embodiments
6-18, wherein D1 and D2 are both --NR6--. [0315] 20. The conjugate
of any one of embodiments 6-19, wherein E1 and E2 are independently
selected from --O-- or --NR6-- or a valence bond. [0316] 21. The
conjugate of any one of embodiments 6-20, wherein E1 is --NR6--.
[0317] 22. The conjugate of any one of embodiments 6-21, wherein E2 is
--NR6--. [0318] 23. The conjugate of any one of embodiments 6-22,
wherein E1 and E2 are both --O--. [0319] 24. The conjugate of any one of
embodiments 6-23, wherein E1 and E2 are both --NR6--. [0320] 25. The
conjugate of any one of embodiments 6-24, wherein W1 through W8
independently are selected from the group consisting of --C(O)NH--,
--NHC(O)--, --C(O)NHCH2--, --CH2NHC(O)--, --C(O)NHS(O)2--,
--S(O)2NHC(O)-- or a valence bond. [0321] 26. The conjugate of any
one of embodiments 6-25, wherein R1, R2, R3, R4 and
R5 independently are selected from hydrogen, --C(O)OH,
--C(O)NH2, --S(O)2OH or C1-6-alkyl; wherein the alkyl
group optionally is substituted with --C(O)OH, --C(O)NH2,
--S(O)2OH. [0322] 27. The conjugate of any one of embodiments 6-26,
wherein --{[(CH2)n1E1]m2-[(CHR2).sub.I2--W3].sub-
.m3}n2-- and [0323] If
{[(CH2)n3E2]m5-[(CHR4).sub.I4--W5]m6}n-
4--, wherein E1 and E2 are 0, are selected from

[0323] ##STR00018## [0324] wherein * is intended to denote a point of
attachment, ie, an open bond. [0325] 28. The conjugate of any one of
embodiments 6-27, wherein A is selected from

##STR00019## ##STR00020##

[0326] wherein * denotes the attachment to B through W. [0327] 29. The
conjugate of any one of embodiments 1-28, wherein the albumin binding
residue via a hydrophilic spacer is attached to the glutamine residue in
the position corresponding to postion 40 in SEQ ID No. 1. [0328] 30. The
conjugate of any one of embodiments 1-29, wherein the albumin binding
residue via a hydrophilic spacer is attached to the glutamine residue in
the position corresponding to postion 141 in SEQ ID No. 1. [0329] 31. The
conjugate of any one of embodiments 1-30, wherein the albumin binding
residue via a hydrophilic spacer is attached to the N-terminal residue of
the growth hormone compound, such as hGH (SEQ ID No. 1). [0330] 32. A
growth hormone conjugate of the formula (II):

[0330] A-W-B-GH-B'-W'-A' (II)

wherein [0331] GH represents a growth hormone compound, [0332] W is a
chemical group linking A and B, [0333] W' is a chemical group linking A'
and B', [0334] A and A' independently represents an albumin binding
residue, [0335] B and B' independently are hydrophilic spacers, and
[0336] pharmaceutically acceptable salts, solvates and prodrugs thereof.
[0337] 33. The conjugate of embodiment 32, wherein GH represents a growth
hormone compound comprising an amino acid sequence having at least 90%
identity to the amino acid sequence of human growth hormone (hGH) (SEQ ID
NO:1). [0338] 34. The conjugate of embodiment 33, wherein GH is hGH (SEQ
ID NO:1). [0339] 35. The conjugate of any one of embodiments 32-34,
wherein W' is selected from W, A' is selected from A and B' is selected
from B. [0340] 36. The conjugate of any one of embodiments 32-35, wherein
W and W', A and A', and B and B' independently are selected from their
respective definitions of any one of embodiments 6-31. [0341] 37. The
conjugate of any one of the previous embodiments, wherein the molar
weight of said hydrophilic spacer is in the range from 80D to 1500D or in
the range from 500D to 1100D. [0342] 38. The conjugate of any one of the
previous embodiments, wherein said albumin binding residue is a
lipophilic residue. [0343] 39. The conjugate of any one of the previous
embodiments, wherein said albumin binding residue binds non-covalently to
albumin. [0344] 40. The conjugate of any one of the previous embodiments,
wherein said albumin binding residue is negatively charged at
physiological pH. [0345] 41. The conjugate of any one of the previous
embodiments, wherein said albumin binding residue has a binding affinity
towards human serum albumin that is below about 10 μM or below about 1
μM. [0346] 42. The conjugate of any one of the previous embodiments,
wherein said albumin binding residue is selected from a straight chain
alkyl group, a branched alkyl group, a group which has an
ω-carboxylic acid group or an ω-carboxylic acid isoster.
[0347] 43. The conjugate of any one of embodiments the previous
embodiments, wherein said albumin binding residue has from 6 to 40 carbon
atoms, from 8 to 26 carbon atoms or from 8 to 20 carbon atoms. [0348] 44.
The conjugate of any one of the previous embodiments, wherein said
albumin binding residue is a peptide, such as a peptide comprising less
than 40 amino acid residues. [0349] 45. The conjugate of any one of the
previous embodiments, wherein said compound is selected from

[0349] ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## [0350] 46. The conjugate of any one of
embodiments 1-45 for use in therapy. [0351] 47. The conjugate of any one
of embodiments 1-45 for use in a method of treating Turner Syndrome;
Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome; chronic
renal disease, juvenile rheumatoid arthritis; cystic fibrosis,
HIV-infection in children receiving HAART treatment (HIV/HALS children);
short children born short for gestational age (SGA); short stature in
children born with very low birth weight (VLBW) but SGA; skeletal
dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature
(ISS); GHD in adults; fractures in or of long bones, such as tibia,
fibula, femur, humerus, radius, ulna, clavicula, matacarpea, matatarsea,
and digit; fractures in or of spongious bones, such as the scull, base of
hand, and base of food; patients after tendon or ligament surgery in e.g.
hand, knee, or shoulder; patients having or going through distraction
oteogenesis; patients after hip or discus replacement, meniscus repair,
spinal fusions or prosthesis fixation, such as in the knee, hip,
shoulder, elbow, wrist or jaw; patients into which osteosynthesis
material, such as nails, screws and plates, have been fixed; patients
with non-union or mal-union of fractures; patients after osteatomia, e.g.
from tibia or 1st toe; patients after graft implantation; articular
cartilage degeneration in knee caused by trauma or arthritis;
osteoporosis in patients with Turner syndrome; osteoporosis in men; adult
patients in chronic dialysis (APCD); malnutritional associated
cardiovascular disease in APCD; reversal of cachexia in APCD; cancer in
APCD; chronic abstractive pulmonal disease in APCD; HIV in APCD; elderly
with APCD; chronic liver disease in APCD, fatigue syndrome in APCD;
Crohn's disease; impaired liver function; males with HIV infections;
short bowel syndrome; central obesity; HIV-associated lipodystrophy
syndrome (HALS); male infertility; patients after major elective surgery,
alcohol/drug detoxification or neurological trauma; aging; frail elderly;
osteo-arthritis; traumatically damaged cartilage; erectile dysfunction;
fibromyalgia; memory disorders; depression; traumatic brain injury;
subarachnoid haemorrhage; very low birth weight; metabolic syndrome;
glucocorticoid myopathy; short stature due to glucucorticoid treatment
inchildren, the acceleration of the healing of muscle tissue, nervous
tissue or wounds; the acceleration or improvement of blood flow to
damaged tissue; or the decrease of infection rate in damaged tissue.
[0352] 48. A pharmaceutical composition comprising a conjugate of any one
of embodiments 1-45, optionally in combination with a pharmaceutical
acceptable excipient. [0353] 49. A method of preparation of a conjugate
of any one of embodiments 1-45, the method comprising introduction of an
albumin binder linker to recombinant produced growth hormone or growth
hormone analogue via the TGase method. [0354] 50. A method of treating
growth hormone deficiency (GHD), the method comprising administrating to
a patient in need thereof an effective amount of a therapeutivcally
effective amount of a conjugate of any one of embodiments 1-45. [0355]
51. A method of treating Turner Syndrome; Prader-Willi syndrome (PWS);
Noonan syndrome; Down syndrome; chronic renal disease, juvenile
rheumatoid arthritis; cystic fibrosis, HIV-infection in children
receiving HAART treatment (HIV/HALS children); short children born short
for gestational age (SGA); short stature in children born with very low
birth weight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia;
achondroplasia; idiopathic short stature (ISS); GHD in adults; fractures
in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna,
clavicula, matacarpea, matatarsea, and digit; fractures in or of
spongious bones, such as the scull, base of hand, and base of food;
patients after tendon or ligament surgery in e.g. hand, knee, or
shoulder; patients having or going through distraction oteogenesis;
patients after hip or discus replacement, meniscus repair, spinal fusions
or prosthesis fixation, such as in the knee, hip, shoulder, elbow, wrist
or jaw; patients into which osteosynthesis material, such as nails,
screws and plates, have been fixed; patients with non-union or mal-union
of fractures; patients after osteatomia, e.g. from tibia or 1st toe;
patients after graft implantation; articular cartilage degeneration in
knee caused by trauma or arthritis; osteoporosis in patients with Turner
syndrome; osteoporosis in men; adult patients in chronic dialysis (APCD);
malnutritional associated cardiovascular disease in APCD; reversal of
cachexia in APCD; cancer in APCD; chronic abstractive pulmonal disease in
APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD,
fatigue syndrome in APCD; Crohn's disease; impaired liver function; males
with HIV infections; short bowel syndrome; central obesity;
HIV-associated lipodystrophy syndrome (HALS); male infertility; patients
after major elective surgery, alcohol/drug detoxification or neurological
trauma; aging; frail elderly; osteo-arthritis; traumatically damaged
cartilage; erectile dysfunction; fibromyalgia; memory disorders;
depression; traumatic brain injury; subarachnoid haemorrhage; very low
birth weight; metabolic syndrome; glucocorticoid myopathy; short stature
due to glucucorticoid treatment inchildren, the acceleration of the
healing of muscle tissue, nervous tissue or wounds; the acceleration or
improvement of blood flow to damaged tissue; or the decrease of infection
rate in damaged tissue, the method comprising administrating to a patient
in need thereof an effective amount of a therapeutivcally effective
amount of a conjugate of any one of embodiments 1-45. [0356] 52. The use
of a conjugate of any one of embodiments 1-45 in the manufacture of a
medicament for the treatment of growth hormone deficiency (GHD). [0357]
53. The use of a conjugate of any one of embodiments 1-45 in the
manufacture of a medicament for the treatment of Turner Syndrome;
Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome; chronic
renal disease, juvenile rheumatoid arthritis; cystic fibrosis,
HIV-infection in children receiving HAART treatment (HIV/HALS children);
short children born short for gestational age (SGA); short stature in
children born with very low birth weight (VLBW) but SGA; skeletal
dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature
(ISS); GHD in adults; fractures in or of long bones, such as tibia,
fibula, femur, humerus, radius, ulna, clavicula, matacarpea, matatarsea,
and digit; fractures in or of spongious bones, such as the scull, base of
hand, and base of food; patients after tendon or ligament surgery in,
e.g., hand, knee, or shoulder; patients having or going through
distraction oteogenesis; patients after hip or discus replacement,
meniscus repair, spinal fusions or prosthesis fixation, such as in the
knee, hip, shoulder, elbow, wrist or jaw; patients into which
osteo-synthesis material, such as nails, screws and plates, have been
fixed; patients with non-union or mal-union of fractures; patients after
osteatomia, e.g., from tibia or 1st toe; patients after graft
implantation; articular cartilage degeneration in knee caused by trauma
or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis
in men; adult patients in chronic dialysis (APCD); malnutritional
associated cardiovascular disease in APCD; reversal of cachexia in APCD;
cancer in APCD; chronic abstractive pulmonal disease in APCD; HIV in
APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome
in APCD; Crohn's disease; impaired liver function; males with HIV
infections; short bowel syndrome; central obesity; HIV-associated
lipodystrophy syndrome (HALS); male infertility; patients after major
elective surgery, alcohol/drug detoxification or neurological trauma;
aging; frail elderly; osteo-arthritis; traumatically damaged cartilage;
erectile dysfunction; fibromyalgia; memory disorders; depression;
traumatic brain injury; subarachnoid haemorrhage; very low birth weight;
metabolic syndrome; glucocorticoid myopathy; short stature due to
glucucorticoid treatment inchildren, the acceleration of the healing of
muscle tissue, nervous tissue or wounds; the acceleration or improvement
of blood flow to damaged tissue; or the decrease of infection rate in
damaged tissue.

[0358] The description herein of any aspect or embodiment of the invention
using terms such as "comprising", "having", "including" or "containing"
with reference to an element or elements is intended to provide support
for a similar aspect or embodiment of the invention that "consists of",
"consists essentially of", or "substantially comprises" that particular
element or elements, unless otherwise stated or clearly contradicted by
context (e.g., a composition described herein as comprising a particular
element should be understood as also describing a composition consisting
of that element, unless otherwise stated or clearly contradicted by
context).

[0359] This invention includes all modifications and equivalents of the
subject matter recited in the aspects or claims presented herein to the
maximum extent permitted by applicable law.

[0360] The present invention is further illustrated by the following
examples which, however, are not to be construed as limiting the scope of
protection. The features disclosed in the foregoing description and in
the following examples may, both separately and in any combination
thereof, be material for realising the invention in diverse forms
thereof.

[0423] The gene coding for the growth hormone compound was inserted
recombinantly into a plasmid vector. A suitable E.coli strain was
subsequently transformed using the plasmid vector. hGH or GH variants may
be expressed with an N-terminal methionine or as a MEAE fusion from which
the MEAE sequence is subsequently cleaved off.

[0424] Cell stock was prepared in 25% glycerol and stored at -80°
C. Glycerol stock strain was inoculated into LB plates and subsequently
incubated at 37° C. overnight. The content of each plate was
washed with LB medium and diluted into 500 mL LB medium for expression.
The cultures were incubated at 37° C. with shaking at 220 rpm
until OD600 0.6 was reached. Succeeding induction was performed
using 0.2 mM IPTG at 25° C. for 6 hrs. Cells were finally
harvested by centrifugation.

[0426] The purification was performed using ion-exchange chromatography
and hydrophibic interaction, followed by removal of the peptide tag using
human dipeptidyl peptidase I (hDPPI) expressed from CHO cell. Final
purification was achieved by isoprecipitation and ion-exchange
chromatography. The purification could also be achieved by using but not
limited to ion-exchange chromatography, hydrophobic interaction
chromatography, affinity chromatography, size exclusion chromatography
and membrane based separation techniques known to a person skilled in the
art.

[0427] The intact purified protein was analysed using MALDI-MS. The
observed mass corresponded to the theoretical mass deduced from the amino
acid sequence. The expected linkage disulfide bonds may be demonstrated
by peptide mapping using trypsin and AspN digestion followed by MALDI-MS
analysis of the digest before and after reduction of the disulfide bonds
with DTT.

Capillary Electrophoresis

[0428] Capillary electrophoresis was carried out using an Agilent
Technologies 3DCE system (Agilent Technologies). Data acquisition and
signal processing were performed using Agilent Technologies 3DCE
ChemStation. The capillary was a 64.5 cm (56.0 cm efficient length) 50
μm i.d. "Extended Light Path Capillary" from Agilent. UV detection was
performed at 200 nm (16 nm Bw, Reference 380 nm and 50 nm Bw). The
running electrolyte was phosphate buffer 50 mM pH 7 (method A). The
capillary was conditioned with 0.1 M NaOH for 3 min, then with Milli-Q
water for 2 min and with the electrolyte for 3 min. After each run, the
capillary was flushed with milli-Q water for 2 min, then with phosphoric
acid for 2 min, and with milli-Q water for 2 min. The hydrodynamic
injection was done at 50 mbar for 4.0 sec. The voltage was +25 kV. The
capillary temperature was 30° C. and the runtime was 10.5 min.

Maldi-Tof Mass Spectrometry

[0429] Molecular weights were determined using the Autoflex Maldi-Tof
instrument (Bruker). Samples were prepared using
alfa-cyano-4-hydroxy-cinnamic acid as matrix.

RP-HPLC

[0430] RP-HPLC analysis was performed on an Agilent 1100 system using a
Vydac 218TP54 4.6 mm×250 mm 5 μm C-18 silica column (The
Separations Group, Hesperia). Detection was by UV at 214 nm, 254 nm, 280
nm and 301 nm. The column was equilibrated with 0.1% trifluoracetic
acid/H2O and the sample was eluted by a suitable gradient of 0 to
90% acetonitrile against 0.1% trifluoracetic acid /H2O.

LC-MS

[0431] LC-MS analysis was performed on a PE-Sciex API 100 or 150 mass
spectrometer equipped with two Perkin Elmer Series 200 Micropumps, a
Perkin Elmer Series 200 auto-sampler, a Applied Biosystems 785A UV
detector and a Sedex 75 Evaporative Light scattering detector. A Waters
Xterra 3.0 mm×50 mm 5μ C-18 silica column was eluted at 1.5
ml/min at room temperature. It was equilibrated with 5% MeCN/0.1%
TFA/H2O and eluted for 1.0 min with 5% MeCN/0.1% TFA/H2O and
then with a linear gradient to 90% MeCN/0.1% TFA/H2O over 7 min.
Detection was by UV detection at 214 nm and Evaporative light Scattering.
A fraction of the column eluate was introduced into the ionspray
interface of a PE-Sciex API 100 mass spectrometer. The mass range
300-2000 amu was scanned every 2 seconds during the run.

Quantification of Protein

[0432] Protein concentrations were estimated by measuring absorbance at
280 nm using a NanoDrop ND-1000 UV-spectrofotometer.

Enzymatic Peptide Mapping for Determination of Site(s) of Derivatization

[0433] Peptide mapping was performed using Asp-N digestion of the reduced
and alkylated protein. First the protein was treated with DTT and
iodoacetamide according to standard procedures. The alkylated product was
purified using HPLC. Subsequently the alkylated purified product was
digested overnight with endoprotease Asp-N (Boehringer) at an
enzyme:substrate ratio of 1:100. The digest was HPLC separated using a
C-18 column and standard TFA/MeCN buffer system. The resulting peptide
map was compared to that of underivatized hGH and fractions with
different retention times were collected and further analyzed using
Maldi-tof mass spectrometry.

SDS Page

[0434] SDS poly-acrylamide gel electrophoresis was performed using NuPAGE
4%-12% Bis-Tris gels (Invitrogen NP0321BOX). The gels were silver stained
(Invitrogen LC6100) or Coomassie stained (Invitrogen LC6065) and where
relevant also stained for PEG with barium iodide as described by M. M.
Kurfurst in Anal. Biochem. 200(2), 244-248, (1992).

Protein Chromatography

[0435] Protein chromatography was performed on an Akta Explorer
chromatographic system and columns from GE Health Care. Anion exchange
was done using a Q-Sepharose HP 26/10 column. Starting buffer was 20 mM
triethanolamine buffer pH 8.5 and eluting buffer was starting buffer +0.2
M NaCl. The compounds were typically eluted with a gradient of 0-75%
eluting buffer over 15 column volumes. De-salting and buffer exchange was
performed using a HiPrep 26/10 column.

[0436] The TGase used in the examples is microbial transglutaminase from
Streptoverticillium mobaraense according to U.S. Pat. No. 5,156,956.

[0438] The BAF-3 cells (a murine pro-B lymphoid cell line derived from the
bone marrow) was originally IL-3 dependent for growth and survival. 11-3
activates JAK-2 and STAT which are the same mediators GH is activating
upon stimulation. After transfection of the human growth hormone receptor
the cell line was turn into a growth hormone-dependent cell line. This
clone can be used to evaluate the effect of different growth hormone
samples on the survival of the BAF-3GHR.

[0440] The cells are washed and re-suspended in starvation medium and
seeded in plates. 10 μl of growth hormone compound or human growth
hormone in different concentrations or control is added to the cells, and
the plates are incubated for 68 hours at 37° C., 5% CO2.

[0441] AlamarBlue® is added to each well and the cells are then
incubated for another 4 hours. The AlamarBlue® is a redox indicator,
and is reduced by reactions innate to cellular metabolism and, therefore,
provides an indirect measure of viable cell number.

[0442] Finally, the metabolic activity of the cells is measure in a
fluorescence plate reader. The absorbance in the samples is expressed in
% of cells not stimulated with growth hormone compound or control and
from the concentration-response curves the activity (amount of a compound
that stimulates the cells with 50%) can be calculated.

Assay for Measuring Rate of Protease Degradation of GH and hGH Compound
Conjugates

[0444] 100 μL of test compound solution at 1 mg/mL in ammonium
bicarbonate buffer is degraded by enzyme for up till 24 hrs at 37°
C. Sub-samples are taken to various time points and the proteolytic
reaction is stopped by acidifying the sample by 10 times dilution into 1%
TFA. These diluted samples are analysed by reversed phase HPLC to
estimate the degree of proteolytic digestion.

HPLC Method:

[0445] 10 μL of the above solution is injected on a reversed phase
Vydac C4 2x150 mm column eluted with a linear gradient from 0.1% TFA in
water to 100% acetonitrile containing 0.1% TFA over a period of 30 min at
a flow rate of 0.2 ml/min. Detection of peaks is performed at 214 nm UV
absorption. Percentage (%) intact compound at time point t=T is
calculated from the peak area at time point t=T (AT) and the peak
area at t=0 (A0) as (AT/A0)×100%. Percentage (%)
intact compound is plotted against time using GraphPad Prims software
ver. 5.01. Half life (T%) is calculated as one phase decay also by
GraphPad Prism software. Examples of enzymes that may be used are
elastase (Sigma from porcine pancrease) and chymotrypsin (Roche
sequencing grade). Example of buffer is 50 mM ammonium bicarbonate,
pH=8.5.

Pharmacokinetics

[0446] The pharmacokinetic of the compounds of the examples is
investigated in male Sprague Dawley rats after intravenous (i.v.) and/or
subcutaneous (s.c.) single dose administration.

[0448] The test compounds are studied in male Sprague Dawley rats weighing
250 g. The test compounds are administered as a single injection either
i.v. in the tail vein or s.c. in the neck with a 25 G needle at a dose of
60 nmol/kg body weight.

[0449] For each test compound blood sampling is conducted according to the
following schedule presented in table 1.

[0450] At each sampling time 0.25 mL blood is drawn from the tail vein
using a 25 G needle. The blood is sampled into a EDTA coated test tube
and stored on ice until centrifugation at 1200×G for 10 min at
4° C. Plasma is transferred to a Micronic tube and stored at
-20° C. until analysis.

[0451] Test compound concentrations are determined by a sandwich ELISA
using a guinea pig anti-hGH polyclonal antibody as catcher, and
biotinylated hGH binding-protein (soluble part of human GH receptor) as
detector. The limit of detection of the assay was 0.2 nM.

[0452] A non-compartmental pharmacokinetic analysis is performed on mean
concentration-time profiles of each test compound using WinNonlin
Professional (Pharsight Inc., Mountain View, Calif., USA). The
pharmacokinetic parameter estimates of terminal half-life (t.sub.%) and
mean residence time (MRT) are calculated.

[0453] In vivo dose-response study in hypophysectomised Sprague Dawley
rats The in vivo dose-response relationship is studied in
hypophysectomised male Sprague Dawley rats. The hypophysectomised rat is
a well known and recognised animal model of growth hormone deficiency,
where no production of growth hormone occurs after the surgical removal
of the pituitary gland. This also leads to low circulating levels of
insulin-like growth factor-1 (IGF-1) another important clinical feature
of growth hormone deficiency in humans.

[0454] The hypophysectomy is performed on 4 week old male rats weighing
90-100 g. The animals entering the study 3-4 weeks after the surgery
weighing 100-110 g. Animals with a body weight gain of more than 10%
during the 3-4 weeks after surgery are not allowed to enter the study.

[0455] Dose response studies are usually performed using five dose levels
of test compound from 1-150 nmol/rat.

Disapperance

[0456] Disapperence rates may be meaussered in pigs, usually at least five
female pigs of crossbred LYD. The pigs are weighted, fasted and issued a
special "pig coat" in order to carry the gamma counter and transmitter
and placed in single pens before the start of the study. All pigs are
fasted for 18 hours prior the study.

[0457] The animals are dosed (60 nmol) subcutaneously on the left and the
right side of the neck respectively with a Novopen3® and a
NovoFine® 28G needle with fixed black needle stopper. Injection depth
is 5 mm. The test solutions (including the compounds) are diluted in a
buffer consisting of: Glycine 20 mg/mL, mannitol 2 mg/mL, NaHCO3 2.4
mg/mL, pH adjusted to 8.2.

[0458] Iodination with 1251 is performed by Chemistry & Isotope Lab.
Novo Nordisk A/S. The final radioactive formulation has a specific
radioactive activity of 3 μCi/mL and is provided in 3 mL Penfills. The
solutions were stored at 2-8° C. until used.

[0459] The desapperence of the radioactive depots is measured by portable
equipment for about 24-48 hours.

Preparation of Albumin Binders

Example 1

Tetrazol OEG Linker (I):

##STR00027##

[0461] The tetrazole OEG linker (I) was synthesised according to scheme 1.

##STR00028## ##STR00029##

[0462] 2 g of Rink-Amide-Resin (2 g, 0.6 mMol/g) was weighed into a flask.
The resin was swelled with NMP (3×30 mL) for 2 hrs.

[0463] Removal of the Fmoc-group: The resin was shaken with 25% piperidine
in NMP (30 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (30 mL) for 1 hour followed by draining and wash with
NMP (6×30 mL).

[0464] Fmoc-Lys(Mtt)-OH and HOBT were weighed into a flask, dissolved in
bromo phenol blue in NMP (30 mL, 0.5 mM). This solution was added to the
drained resin above followed by addition of DIC. The reaction was shaken
at ambient temperature for 21 hrs. The resin was drained and washed with
NMP (6×30 mL) followed by washing with DCM (3×30 mL).

[0465] The resin was treated with hexafluorisopropanol (20 mL) for 10 min.
Shaken for 10 min. The resin was drained and washed with DCM (3×30
mL). The resin was treated with hexafluorisopropanol (20 mL) for 10 min
again and shaked for 10 min. The resin was drained and washed with DCM
(3×30 mL) followed by drained and washed with NMP (3×30 mL).

[0466] Boc-4-ABZ-OH and HOBT were weighed into a flask, dissolved in bromo
phenol blue in NMP (30 mL, 0.5 mM). This solution was added to the
drained resin above followed by addition of DIC. The reaction was shaken
at ambient temperature. The resin was drained and washed with NMP
(6×30 mL).

[0467] Removel of the Fmoc-group: The resin was shaken with 25% piperidine
in NMP (10 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (10 mL) for 1 hour followed by draining and wash with
NMP (6×15 mL).

[0468] Fmoc-OEG-OH and HOBT were weighed into a flask, dissolved in brom
phenol blue in NMP (15 mL, 0.5 mM). This solution was added to the
drained resin followed by addition of DIC. The reaction was shaken at
ambient temperature for 23 hrs. The resin was drained and washed with NMP
(6×15 mL).

[0469] Removal of the Fmoc-group: The resin was shaken with 25% piperidine
in NMP (10 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (10 mL) for 1 hour followed by draining and wash with
NMP (6×15 mL).

[0470] Fmoc-Glu-O-t-Bu and HOBT were weighed into a flask, dissolved in
bromo phenol blue in NMP (15 mL, 0.5 mM). This solution was added to the
drained resin followed by addition of DIC. The reaction was shaken at
ambient temperature for 18 hrs. The resin was drained and washed with NMP
(6×15 mL).

[0471] Removal of the Fmoc-group: The resin was shaken with 25% piperidine
in NMP (10 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (10 mL) for 1 hour followed by draining and wash with
NMP (6×15 mL).

[0472] Fmoc-Glu-O-t-Bu and HOBT were weighed into a flask, dissolved in 15
ml 0.5 mM bromo phenol blue in NMP. This solution was added to the
drained resin followed by addition of DIC. The reaction was shaken at
ambient temperature for 18 hrs. The resin was drained and washed with NMP
(6×15 mL).

[0473] Removal of the Fmoc-group: The resin was shaken with 25% piperidine
in NMP (10 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (10 mL) for 1 hour followed by draining and washing
with NMP (6×15 mL).

[0474] Fmoc-OEG-OH and HOBT were weighed into a flask, dissolved in bromo
phenol blue in NMP (15 mL, 0.5 mM). This solution was added to the
drained resin followed by addition of DIC. The reaction was shaken at
ambient temperature. The resin was drained and washed with NMP
(6×15 mL).

[0475] Removal of the Fmoc-group: The resin was shaken with 25% piperidine
in NMP (10 mL) for 10 min. The resin was drained and treated with 25%
piperidine in NMP (10 mL) for 1 hour followed by draining and washing
with NMP (6×15 mL).

[0476] 4-(16-1H-Tetrazol-5-yl-hexadecanoylsulfamoyl)-butyric acid and HOBT
were weighed into a flask, dissolved in bromo phenol blue in NMP (15 mL,
0.5 mM). This solution was added to the drained resin followed by the
addition of DIC. The reaction was shaken at ambient temperature for 21
hrs. The resin was drained and washed with NMP (6×15 mL NMP)
followed by draining and wash with DCM (6×15 mL).

[0477] The resin was cleaved with a mixture of 95% TFA in water (10
mL)+DCM (0,25 mL) and TIPS (0.25 mL). The resin was shaken for 2 hrs at
ambient temperature and filtered into cold diethyl ether (75 mL). The
resulting precipitate was isolated by centrifugation followed by washing
with diethyl ether (3×) and dried in vacuum for 48 hrs affording
crude 300 mg of the compound. TOF-MS: Rt=4,7 min, mass 1268.71

[0478] The crude compound was purified on prep-HPLC (GILSON). T2145-10;
30->80% MeCN. Pooled fractions were evaporated to dryness on rotavap
and the residue dissolved in H2O/MeCN 1:1 and freezedried over night
affording 170 mg of the compound.

Example 2

[0479] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Lys(Mtt)-OH and Wang Resin.

##STR00030##

Example 3

[0480] In a similar way as described in Example 1 above and depicted below
the following compound was prepared using Boc-Gly-PAM resin as starting
material.

##STR00031##

Example 4

[0481] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00032##

Example 5

[0482] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00033##

Example 6

[0483] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00034##

Example 7

[0484] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00035##

Example 8

[0485] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00036##

Example 9

[0486] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Wang Resin.

##STR00037##

Example 10

[0487] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00038##

Example 11

[0488] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00039##

Example 12

[0489] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Wang Resin.

##STR00040##

Example 13

[0490] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00041##

Example 14

[0491] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00042##

Example 15

[0492] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00043##

Example 16

[0493] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00044##

Example 17

[0494] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00045##

Example 18

[0495] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00046##

Example 19

[0496] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00047##

Example 20

[0497] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00048##

Example 21

[0498] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Glu(ODmab)-OH and 2-chlorotrityl
chloride resine.

##STR00049##

Example 22

[0499] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Glu(ODmab)-OH and 2-chlorotrityl
chloride resine.

##STR00050##

Example 23

[0500] In a similar way as described in Example 1 above the following
compound was prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00051##

Example 24

[0501] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

##STR00052##

Example 25

[0502] In a similar way as described in Example 1 above the following
compound may be prepared using FMOC-Glu(ODmab)-OH and 2-chlorotrityl
chloride resine.

[0504] The use of a transglutaminase to attach an aldehyde handle to GH on
glutamine residues has previously been described in WO2005/070468. The
method may be used in accordance with the present invention for
attachment of a albumin binder based linker of formula A-W-B1-NH2. The
TGase used is microbial transglutaminase from Streptoverticillium
mobaraense according to U.S. Pat. No. 5,156,956.

[0505] The reaction may be performed as follows: GH (I) is initially
transaminated with 1,3-diamino-2-propanol (II) as described in
WO2005/070468:

##STR00054##

[0506] In the next step, transaminated GH (III) is added periodate. The
oxidation is typically done at low temperature, such as 4-10° C.
over 30 min. optionally in the dark. Periodate may oxidize metheonine
residues in GH to their corrosponding metheonine sulfoxide residues. To
minimize this oxidation risk, small molecule organic thioethers may be
added during periodate oxidation. A suitable organic thioether is
3-methylthiopropan-1-ol but the skilled person will be able to suggest
others.

Oxidation of Transaminated GH Compound (Ill):

##STR00055##

[0507] Buffer change may be performed in order to obtain an acid solution
required for efficient sodium cyano borohydride reduction. Typically, an
excess of A-W-B1-NH2 amine is used, and sodium cyanoborohydride may be
added in smaller portions over time. Reductive amination of (IV) with
albumin binder linker (V):

##STR00056##

The later reaction may be performed as follows:

[0508] A solution of oxidized transaminated GH (IV) is added a solution of
albumin binder linker (V) in a mixture of AcOH (1,5 mL) and 50 mM MES
(0.5 mL) at pH 6.00. The resulting reaction mixture is gently shaken at
RT for 30 min. at which time a NaCNBH3 solution (15 μL, (22 mg
NaCNBH3 dissolved in 500 μL Milli-Q water+AcOH (15 μL))) is
added. The sample is covered with tin foil and stirrer over night at RT.

[0509] The conjugate can be isolated by anion exchange chromatography as
follows: Acetic acid is removed by buffer changed with pure water
(3×) using Amicon Ultra15 devices (Ultracel 10K tubes) by
centrifugation at 4000 rpm/min. for 3×8 min. The mixture is then
buffer changed to 20 mM TEA, pH: 8.50 using Amicon Filter devises and
diluted to a final volume of 50 mL with 20 mM TEA, before loading it on a
HiLoad Q Sepharose, 26/10 column. The column is initially washed with 20
mM TEA, pH 8.50 (buffer A) and then eluted with 20 mM TEA, 500 mM NaCl,
pH 8.50 (buffer B) using a 0-100%(B) gradient over 20 CV, with a flow
rate of 2 ml/min. The pooled fractions were buffer changed 5 times to 10
mM ammoniumbicarbonate buffer in pure water using Amicon Ultra15 devices
(Ultracel 10K tubes) by centrifugation at 4000 rpm/min. for 3×8 min

Example 27

[0510] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 2.

##STR00057##

Example 28

[0511] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 4.

##STR00058##

Example 29

[0512] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 5.

##STR00059##

Example 30

[0513] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 6.

##STR00060##

Example 31

[0514] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 7.

##STR00061##

Example 32

[0515] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 8.

##STR00062##

Example 33

[0516] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 9.

##STR00063##

Example 34

[0517] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 10.

##STR00064##

Example 35

[0518] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 11.

##STR00065##

Example 36

[0519] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 12.

##STR00066##

Example 37

[0520] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 13.

##STR00067##

Example 38

[0521] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 14.

##STR00068##

Example 39

[0522] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 15.

##STR00069##

Example 40

[0523] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 16.

##STR00070##

Example 41

[0524] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 17.

##STR00071##

Example 42

[0525] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 18.

##STR00072##

Example 43

[0526] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 19.

##STR00073##

Example 44

[0527] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 20.

##STR00074##

Example 45

[0528] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 21.

##STR00075##

Example 46

[0529] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 22.

##STR00076##

Example 47

[0530] In a similar way as described in Example 26 above the following
compound was prepared using albumin binder from Example 23.

##STR00077##

Example 48

[0531] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 24.

##STR00078##

Example 49

[0532] In a similar way as described in Example 26 above the following
compound may be prepared using albumin binder from Example 25.

[0534] The use of a transglutaminase to attach an aldehyde handle to GH on
glutamine residues has previously been described in WO2005/070468. The
method may be used in accordance with the present invention for
attachment of a albumin binder based linker of formula A-W-B1-NH2. The
TGase used is microbial transglutaminase from Streptoverticillium
mobaraense according to U.S. Pat. No. 5,156,956.

[0535] The reaction may be performed as follows: GH (I) is initially
transaminated with 1,3-diamino-2-propanol (II) as described in
WO2005/070468:

[0536] Transaminated hGH Gln40 (Ill) was obtained from Example 26 as
a biproduct from the CIE chromatography purification.

##STR00080##

[0537] In the next step, transaminated GH (III) is added periodate. The
oxidation is typically done at low temperature, such as 4-10° C.
over 30 min. optionally in the dark. Periodate may oxidize metheonine
residues in GH to their corrosponding metheonine sulfoxide residues. To
minimize this oxidation risk, small molecule organic thioethers may be
added during periodate oxidation. A suitable organic thioether is
3-methylthiopropan-1-ol but the skilled person will be able to suggest
others.

[0538] Oxidation of transaminated GH compound (III):

##STR00081##

[0539] Buffer change may be performed in order to obtain an acid solution
required for efficient sodium cyano borohydride reduction. Typically, an
excess of A-W-B1-NH2 amine is used, and sodium cyanoborohydride may be
added in smaller portions over time.

Reductive amination of (IV) with albumin binder linker (V):

##STR00082##

The later reaction may be performed as follows:

[0540] A solution of oxidized transaminated GH (IV) is added a solution of
bile acid linker (V) in a mixture of AcOH (1.5 mL) and 50 mM MES (0.5 mL)
at pH 6.00. The resulting reaction mixture is gently shaken at RT for 30
min. at which time a NaCNBH3 solution (15 μL, (22 mg NaCNBH3
dissolved in 500 μL Milli-Q water+AcOH (15 μL))) is added. The
sample is covered with tin foil and stirrer over night at RT.

[0541] The conjugate can be isolated by anion exchange chromatography as
follows: Acetic acid is removed by buffer changed with pure water (3X)
using Amicon Ultra15 devices (Ultracel 10K tubes) by centrifugation at
4000 rpm/min. for 3×8 min. The mixture is then buffer changed to 20
mM TEA, pH: 8.50 using Amicon Filter devises and diluted to a final
volume of 50 mL with 20 mM TEA, before loading it on a HiLoad Q
Sepharose, 26/10 column. The column is initially washed with 20 mM TEA,
pH 8.50 (buffer A) and then eluted with 20 mM TEA, 500 mM NaCl, pH 8.50
(buffer B) using a 0-100%(B) gradient over 20 CV, with a flow rate of 2
mL/min. The pooled fractions were buffer changed 5 times to 10 mM
ammoniumbicarbonate buffer in pure water using Amicon Ultra15 devices
(Ultracel 10K tubes) by centrifugation at 4000 rpm/min. for 3×8 min

[0542] In a similar way as described in Example 50 above the following
compound was prepared using albumin binder from Example 6.

[0546] hGH (23 mg) was dissolved in Hepes buffer (2.3 mL 0.25 mM pH 7,0).
C20diacid-Trx-yGlu-Glu-OEG-OEG-Gly-Gly-dimethylacetal (2 mg, see
example 3 above) was treated with TFA (50 μL) for 6 min. and
evaporated to dryness in vacuum. The residue was stripped with EtOH (200
μL) and evaporated to dryness in vacuum. The residue was dissolved in
DMF (100 μL) and added to the hGH solution. A precipitate was formed
and redissolved by addition of DMF (1 mL). After 1 hr a solution of
NaCNBH3 (20 mg, in 0.5 mL MeCN (230 μL)) was added portionwise
and left for 20 hrs. The reaction was quenched by addition of AcOH (2 mL)
and diluted with water to a total volume of 20 ml and purified on prep.
HPLC on a C18 column with a gradient of MeCN/0.1% TFA from 40-80% against
0.1% TFA in water. The latest eluting peak was collected, diluted from
70% MeCN to 10% with water and lyophilized affording 4,51 mg of the
compound. TOF-MS: mass 23.237,6

Example 52

[0547] GH compounds as described herein were tested in one or more of the
assays described herein above.

[0548] EC50 values based on BAF assay as well as half life (T%) and mean
residence time (MRT) were determined and included in table 2 below.

[0549] Disapperence rate for selected compounds were meaussered in five
female pigs of crossbred LYD as described above. The result are presented
as AUC (0-45 hrs) in table 2. The solubility enhancing propertiy
expressed as cLogP of the space (B) as determined by the method described
above is also included in table 2.

[0550] Albumin binding affinity as determined by Biacor and or HPLC is
also included in table 2.

[0551] Data relating to a pegylated hGH compound is included as well.
PEG-hGH being identical to the compound disclosed in Example 1 of
WO2006024953A2.